High affinity vegf-receptor antagonists

ABSTRACT

A cell-based screen is reported can be used to identify specific receptor-binding compounds in a combinatorial library of peptoids (N-alkylglycine oligomers) displayed on beads. This strategy was applied to the isolation of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2)-binding peptoids, which were optimized to create lead compounds with high affinity for VEGFR2. One of these peptoids was shown to be an antagonist of VEGF-VEGFR2 interaction and receptor function.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/948,845, filed Jul. 10, 2007, the entire contents of whichare expressly incorporated herein by reference.

This invention was made with government support under N01-HV28185awarded by National Heart, Lung and Blood Institute. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to fields of peptide and peptoidchemistry, molecular biology and cell biology. More particularly, thepresent invention relates to peptidomimetics that antagonize the VEGFreceptor and inhibit angiogenesis as well as methods with which toidentify said antagonists. In particular, such inhibitors may find usein the treatment of diseases such as cancer and macular degeneration.

2. Description of Related Art

Monoclonal antibodies that antagonize the formation of various hormonereceptor interactions are used widely in the clinic. For example,Remicade, and Adalimumab are anti-arthritic antibodies that blockbinding of Tumor Necrosis Factor (TNF)-α to its cognate receptor andthus reduce inflammation (Taylor, 2003). Avastin (Gerber and Ferrara,2005), a Vascular Endothelial Growth Factor (VEGF)-binding antibody,prevents VEGF from docking with VEGF Receptor 2 (VEGFR2) (Hicklin andEllis, 2005), a key step in the angiogenic cascade (Folkman, 1990) thatis critical to support solid tumor survival and proliferation (Dvorak,2002; Millauer et al., 1993; Zeng et al., 2001). Avastin is employedwidely in the treatment of various tumors (Hurwitz et al., 2004; Johnsonet al., 2004) and, more recently, to block blood vessel formation in“wet” macular degeneration (Ambresin and Mantel, 2007). Whiletherapeutic monoclonal antibodies are obviously of great value, they arenot without drawbacks. They are relatively difficult and expensive tomanufacture in large quantities, there is some problem with immunereactions (Stevenson, 2005), though this has been minimized byhumanization (Mateo et al., 2000).

Thus, it would be of great interest to develop relatively small, easilymanipulable synthetic compounds that display antibody-like affinity andspecificity for a given receptor, but which could be made cheaply and beeasily tailored to carry cargo of various sorts. Unfortunately, it hastraditionally proven difficult to isolate small molecule antagonists ofprotein-protein interactions, particularly those involving large,shallow interaction surfaces typical of hormone receptor complexes(Whitty and Kumaravel, 2006). While many examples are known of smallmolecules that act as agonists or antagonists of integral membranereceptors, these generally act by alternative mechanisms, for example asinhibitors of the ligand-activated kinase activity of the receptor(Cabebe and Wakelee, 2006; Ciardiello et al., 2003; Thomas, 2003). Onthe other hand, there are several examples of receptor- orhormone-binding peptide antagonists (D'Andrea et al., 2006). This is notsurprising, since peptides are much better able to mimic the naturalbinding partner of a hormone or receptor than is a classical smallmolecule. In a few cases, these peptide antagonists have been developedinto clinically useful compounds (Liu et al., 2007) but this approach todrug development is severely limited by the sensitivity of peptides toproteolysis. However, there remains a need in the field to developagents that are able to antagonize those proteins involved withangiogenesis, particularly in the context of pathologic conditions thatrequire the development of new blood vessels.

SUMMARY OF THE INVENTION

Thus, in accordance with the present invention, there is provided amethod of antagonizing VEGFR, in particular VEGFR2, comprisingcontacting a cell containing the receptor with a compound having theformula:

The cell may be an endothelial cell, such as a vascular endothelialcell. The compound may comprise a dimer of the formula shown above,where two monomers are connected by a linker, as exemplified below:

wherein W is defined as can be any arrangement of from 1-1000 carbon,hydrogen, nitrogen, sulfur, oxygen, chlorine, bromine, fluorine orsilicon atoms, including, but not limited to biotin, fluorescein orother fluorescent molecules or any commonly used detectable moiety.Other linkers may also be utilized, and the foregoing linker is purelyexemplary. The compound may be formulated in a lipid vehicle.

The cell may be located in a subject with cancer, and the vascularendothelial cell may be tumor-related, i.e., associated with thedevelopment of blood vessels feeding a solid tumor. The cancer may be aglioma, a sarcoma, or a myeloma. The cancer may also be a lung cancer, askin cancer, a head & neck cancer, a stomach cancer, a breast cancer, acolon cancer, a pancreatic cancer, a liver cancer, an ovarian cancer, auterine cancer, a cervical cancer, a testicular cancer, a rectal cancer,an esophageal cancer, or a brain cancer. The cancer may be recurrent,metastatic or multi-drug resistant. The subject may be furtheradministered a chemotherapeutic, radiotherapeutic, immunotherapeutic oranti-cancer gene therapy. The compound may be delivered intravenously,intraarterially, subcutaneously, intra-tumorally, orally, by nasalinhalation, or any other suitable route given the location of the cancercell.

The cell also may be located in a subject with a non-cancerhyperproliferative state, such as macular (wet) degeneration. Suchtreatments will generally follow those provided above for cancer, exceptthat delivery may involve local administration to areas such as the eye,for example, by injection or by topical opthalmic solution. Combinationtherapies, using agents that are used to treated these other diseasesalso are contemplated.

Also provided is a pharmaceutical formulation comprising a compoundhaving the structure:

dispersed in a pharmacologically acceptable medium, diluent orexcipient. The compound may comprise a homodimer of the formula shownabove, where two monomers are connected by a linker, or a heterodimer ofCompound I and Compound III, connected by a linker:

The pharmacologic formulation may comprise a lipid formulation.

In yet another embodiment, there is provided a method of inhibiting VEFGsignaling comprising contacting a cell expressing a VEGFR2 with acompound having the general formula:

wherein X, Y and Z are defined as shown above and R can be anyarrangement of from 1-1000 carbon, hydrogen, nitrogen, sulfur, oxygen,chlorine, bromine, fluorine or silicon atoms, including, but not limitedto H and CH₃.

The compound may comprise a dimer of the formula shown above, where twomonomers are connected by a linker. R, X, Y and Z would be defined asabove in Formula I, and W can be any arrangement of from 1-1000 carbon,hydrogen, nitrogen, sulfur, oxygen, chlorine, bromine, fluorine orsilicon atoms, including, but not limited to biotin, fluorescein orother fluorescent molecules. Other linkers may also be utilized, and theforegoing linker is purely exemplary. Moreover, the dimer may be ahomodimer comprised of identical monomeric units linked together (i.e.,R, X, Y and Z are identical in the two linked units) or could be aheterodimer of two different molecules with non-identical R, X, Y, and Zgroups. The atoms linking the two units of Formula II shown below areexemplary in nature, and other linker arms are not excluded:

The compound may be formulated in a lipid vehicle. The cell may be anendothelial cell, such as a vascular endothelial cell. The human subjectis contacted with said compound more than once The cell may be locatedin a human subject, for example, one that suffers from glioma, sarcomaor myeloma. Alternatively, the subject may suffer from lung cancer, skincancer, head & neck cancer, stomach cancer, breast cancer, colon cancer,pancreatic cancer, liver cancer, ovarian cancer, uterine cancer,cervical cancer, testicular cancer, rectal cancer, esophageal cancer, orbrain cancer. The compound may be formulated in a lipid vehicle. Thehuman subject may further be treated with chemotherapeutic,radiotherapeutic, immunotherapeutic or anti-cancer gene therapy, such asa chemotherapeutic, radiotherapeutic, immunotherapeutic or anti-cancergene therapy. The cancer may be recurrent, metastatic, or multi-drugresistant.

The human subject may alternatively suffer from a non-cancer diseasecharacterized by abnormal or pathologic angiogenesis, such as macular(wet) degeneration. The human subject may be further treated with asecond therapy for said non-cancer disease state.

In still yet another embodiment, there is provided a pharmaceuticalformulation comprising a compound having the structure:

dispersed in a pharmacologically acceptable medium, diluent orexcipient. R can be any arrangement of from 1-1000 carbon, hydrogen,nitrogen, sulfur, oxygen, chlorine, bromine, fluorine or silicon atoms,including, but not limited to H and CH₃ The compound may be a dimer ofFormula I, said dimer comprising a linker that replaces the free —OH or—NH₂ group of each monomer. The compound may be a homodimer, or may be aheterodimer of two different compounds of Formula I, said homo- orheterodimer comprising a linker that replaces the —OH or —NH group ofeach monomer. The formulation may comprise a lipid formulation. Thecompound may have the structure:

The compound may also have the structure:

wherein R, W, Y and Z are defined as above.

There is also provided a method by which to identify molecules in alibrary that bind to a molecule displayed on the surface of a cell. Thismethod is distinguished from existing protocols in that it demands thatthe isolated ligand have extremely high specificity for the targetreceptor.

As shown in FIG. 1, the method involves screening a library of compoundsdisplayed on beads against a mixture of two cell types. The two celltypes are identical except for the presence or absence of the targetreceptor (VEGFR2 in the case of FIG. 1). This is achieved by beginningwith a cell type that does not naturally express the target receptor.These cells are labeled with a quantum dot or other appropriate dye of agiven color. The target receptor is then expressed in this same celltype. The gene for the target receptor can be introduced in manydifferent ways, for example by transfection, by virus-mediatedintegration or other methods known to those skilled in the art. Thecells expressing the target receptor are labeled with a differentcolored quantum dot or appropriate dye. The two cell types, that differsolely in the presence or absence of the target receptor, are then mixedtogether. The mixed population is then treated so as to de-adhere thecells from the plate without destroying the cell surface receptors(i.e., proteolysis is avoided) and the “free” cells are incubated withthe bead library. After a suitable incubation and washing, the beads areexamined so as to distinguish those that bind ONLY the cells containingthe target receptor and that do not bind the cells lacking the targetreceptor. This means that if the molecule displayed on the bead iscapable of binding to any other molecule on the surface of the cellbesides the target receptor, it will bind both red and green cells andthus be rejected as a potential hit.

The cells employed in this screen can be any cell that lacks the targetreceptor. This includes T cells, B cells, epithelial cells, kidney cellsor any other culturable cell type. It also includes single celledorganisms such as yeast or Eschericia coli. The target receptor may beany protein that exposes part of its structure on the cell surface so asto be accessible to cell impermeable molecules.

The method described may employ two different cells, one carrying thetarget receptor, the other not, labeled with two different colored dyesor may be carried out with multiple cells carrying different receptorsand each labeled with a different colored dye. Such a multi-color assaywould be useful in identifying compounds capable of distinguishingbetween very closely related receptors, for example VEGFR1 and VEGFR2(see FIG. 24).

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

These, and other, embodiments of the invention will be betterappreciated and understood when considered in conjunction with thefollowing description and the accompanying drawings. It should beunderstood, however, that the following description, while indicatingvarious embodiments of the invention and numerous specific detailsthereof, is given by way of illustration and not of limitation. Manysubstitutions, modifications, additions and/or rearrangements may bemade within the scope of the invention without departing from the spiritthereof, and the invention includes all such substitutions,modifications, additions and/or rearrangements.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-D. A two-color, cell-based assay for the identification ofpeptoid ligands for VEGFR2. (FIG. 1A) Schematic representation of theassay. The large blue circles represent peptoid library beads, the smallred and green circles represent quantum dot-stained PAE/KDR (VEGFR2 overexpressing) cells and PAE parental (lacking VEGFR2) cells, respectively.Beads that display peptoids that bind specifically to VEGFR2 shouldretain only red-stained cells. (FIG. 1B) Structure of the peptoidlibrary employed in the screen, Top: general structure of all of thecompounds inn the library (attached to bead via their C-terminalcarboxyl group). Three residues (two Lys, and one Leu) at C-terminuswere fixed and the remaining six residues (drawn in blue) werediversified (side chains represented by “R”, drawn in red). Box: theamines employed to make the library. The nitrogen shown in blue becomesthe main chain nitrogen in the peptoid. (FIG. 1C) and (FIG. 1D)Fluorescence microscopic images of select beads after screening andwashing (10× magnification; DAPI filter). The arrows in (FIG. 1C)indicate beads that bind both cells that do and do not express VEGFR2.The bead indicated by the arrow in (FIG. 1D) represents one of five outof ≈300,000 observed to bind only red-stained cells.

FIG. 2. Chemical structures of identified ‘hit’ compounds from on-beadcell screening GU40A. Nlys-Nmea-Nleu-Nall-Ntrp-Nlys-Nleu-Nlys-Nlys;GU40B: Nlys-Npip-Nleu-Nlys-Npip-Nlys-Nleu-Nlys-Nlys; GU40C:Nlys-Nleu-Nlys-Nmba-Npip-Nlys-Nleu-Nlys-Nlys; GU40D:Nlys-Nlys-Ntrp-Npip-Nleu-Nall-Nleu-Nlys-Nlys; GU40E:Ntrp-Nlys-Nffa-Nlys-Nleu-Nlys-Nleu-Nlys-Nlys

FIGS. 3A-B. The peptoids GU40C and GU40E are VEGFR2 ligands. (FIG. 3A)Binding isotherms of fluoreceinated peptoids GU40C and GU40E againstimmobilized VEGFR2, CD105 and GST proteins evaluated by ELISA-like assayusing immobilized. Data points represent mean of duplicate measurementswith error bars corresponding to the standard error of the mean. (FIG.3B) Binding competition of fluoresceinated GU40C and GU40E peptoids withcontrol peptoid to VEGFR2 evaluated by ELISA-like assay. Both peptoidswere at 1 μM and control peptoid was at 10 μM. No significant disruptionof the selected peptoid-VEGFR2 complexes by the control peptoid wasobserved. Data points represent the mean of duplicate measurements witherror bars corresponding to the standard error of the mean.

FIGS. 4A-D. Dimeric peptoid design and binding evaluation. (FIG. 4A)General chemical structure of the dimeric compounds tested. Longerdimeric compounds (GU40C1-5 or GU40E 1-5) contain two full monomericunits, connected by a Lys (blue) and a variable number of γ-aminobutyricacid (red) or γ-aminohexanoic acid (brown) linkers (refer to Table 2).The shorter dimers lack the γ-aminobutyric acid and γ-aminohexanoic acidlinkers as well as one to three of the fixed residues at the C-terminusof each monomer unit (refer to Table 2). In these shorter dimers, twotruncated monomer units were directly connected via Lys residue. Eachcompound was synthesized with a C-terminal Cys residue that was used toattach Fluorescein via maleimide chemistry. (FIG. 4B) Binding affinities(shown as dissociation constants; K_(D)) for the two series ofhomo-dimeric peptoids derived from monomers GU40C and GU40E. (FIG. 4C)Chemical structure of the best dimeric ligand identified; GU40C4. (FIG.4D) Binding isotherm of fluoresceinated GU40C4 for immobilized VEGFR2extracellular domain (K_(D)≈30 nM). Data points represent the mean offour measurements with error bars corresponding to the standard error ofthe mean.

FIGS. 5A-H. Cell specific binding study for GU40C4. All the cells weretreated with biotinylated GU40C4 and subsequently withstreptavidin-conjugated red quantum dots except for (FIG. 5C), which wastreated only with streptavidin-conjugated red quantum dot as a control.The nuclei are stained with DAPI. Fluorescence microscopic images weretaken under DAPI filter at 40× magnification. (FIG. 5A) PAE/KDR cells;(FIG. 5B) PAE/KDR cells+100-fold excess of unlabeled GU40C4; (FIG. 5C)PAE/KDR cells; (FIG. 5D) PAE parental cells; (FIG. 5E) Hela cells; (FIG.5F) HEK-293 cells; (FIG. 5G) MCF-7 cells; (FIG. 5H) Human foreskinfibroblast (HFF) cells.

FIGS. 6A-C. Effects of GU40C4 on VEGF-induced, VEGFR2-dependentfunctions. (FIG. 6A) Effect of the indicated levels of GU40C4 andAvastin on autophosphorylation of VEGFR2 in PAE/KDR cells in thepresence and absence of VEGF (1.3 nM) using antibodies for VEGFR2 andphosphorylated VEGFR2. (FIG. 6B) Quantification of the data shown in(FIG. 6A) reveal an IC₅₀ value of 0.9 μM. The data represent the averageof four independent experiments with the error bars corresponding to thestandard error of the mean. (FIG. 6C) Quantification of HUVECproliferation in the absence and presence of 1.3 nM VEGF, competing withGU40C4. After four days, viable cells were determined using aluminescent assay. HUVEC were treated as follows (starting from left),without VEGF, VEGF only, VEGF+0.1 μM GU40C4, VEGF+1 μM GU40C4, VEGF+10μM GU40C4, VEGF+10 μM FLAG peptide (control). GU40C4 was able to inhibitHUVEC proliferation halfway at 1 μM and almost to the basal level at 10μM. FLAG peptide had no effect on HUVEC proliferation at 10 μM. Datapoints represent the mean of duplicate measurements with error barscorresponding to the standard error of the mean.

FIGS. 7A-C. Effects of GU40C4 on VEGF-induced tube formation in HUVECcells. HUVECs were grown on endothelial cell medium gel, and weretreated with 1.3 nM VEGF and different competing amounts of GU40C4, or10 μM control peptoid, GU40C (monomer) and GU40CC (ineffective dimer).(1) without VEGF treatment, (2) VEGF only, (3) VEGF+0.1 μM GU40C4, (4)VEGF+1 μM GU40C4, (5) VEGF+10 μM GU40C4, (6) VEGF+10 μM control peptoid,(7) VEGF+10 μM GU40C (monomer), (8) VEGF+10 μM GU40CC (ineffectivedimer). GU40C4 was able to disrupt HUVEC tube formation at 1 μM and 10μM concentrations. Control peptoid and GU40CC (ineffective dimer) had nodetectable effect on HUVEC tube formation at 10 μM. GU40C (monomer) hada small effect. Images were taken at 10× magnification and eachexperiment was repeated three times.

FIG. 8. Quantification of the % phosphorylation of above western blotanalysis of three monomeric peptoids GU40B, GU40C and GU40E in PAE/KDRcells under 1.3 nM VEGF induction. All three peptoids were used at 500μM concentration. Peptoid GU40B had no effect while GU40C and GU40Eantagonized VEGFR2 phosphorylation by approximately 75%.

FIG. 9. Western blot analysis of monomers affecting on VEGF inducedVEGFR2 phosphorylation. Western blot analysis of three monomericpeptoids (1: GU40B, 2: GU40C, 3: GU40E) to detect VEGFR2 phosphorylationlevels in PAE/KDR cells under 1.3 nM VEGF induction. All three peptoidswere used at 500 μM concentration. None of the peptoids exhibitedagonist activity. GU40C and GU40E were weak antagonists.

FIGS. 10A-B. (FIG. 10A) Structure of the control peptoid(Nmea-Nmea-Nser-Nmea-Nmea-Nser-Nmea-Nser-Nser). (FIG. 10B) Sequence andstructure of FLAG peptide used as the control ligand in HUVECproliferation assay.

FIG. 11. Binding isotherm of GU40C4 derived from FluorescencePolarization. Binding isotherm of GU40C4 derived from fluorescencepolarization experiments. 0.5 nM fluoresceinated GU40C4 was treated withincreasing amounts of soluble VEGFR2. K_(D)≈20 nM.

FIG. 12. HUVEC Tube formation assay; GU40C4 inhibits VEGF-induced tubeformation in vitro. HUVECs were grown on endothelial cell medium gel,and treated with 1.3 nM VEGF and different amounts of GU40C4, or 10 μMcontrol peptoid, GU40C (monomer) or GU40CC (ineffective dimer).(Panel 1) without VEGF treatment, (Panel 2) VEGF only, (Panel 3)VEGF+0.1 μM GU40C4, (Panel 4) VEGF+1 μM GU40C4, (Panel 5) VEGF+10 μMGU40C4, (Panel 6) VEGF+10 μM control peptoid, (Panel 7) VEGF+10 μM GU40C(monomer), (Panel 8) VEGF+10 μM GU40CC (ineffective dimer). GU40C4disrupted HUVEC tube formation at 1 μM and 10 μM concentrations. Controlpeptoid and GU40CC (ineffective dimer) had no detectable effect on HUVECtube formation at 10 μM. GU40C (monomer) had a small effect. Images weretaken at 100× total magnification and each experiment was repeated threetimes.

FIG. 13. Annotated Structure of GU40C. Residues are numbered startingfrom C-terminus.

FIG. 14. Glycine (black bars) sarcosine (grey bars) scan binding resultsof GU40C. Please refer FIG. 13 for residue numbers.

FIGS. 15A-B. Shortest derivative of GU40C which contains only theimportant side chains and it's binding isotherm. (FIG. 15A) Structure ofGU40C(1) (FIG. 15B) Binding isotherms of GU40C, GU40(1) and a controlpeptoid that does not selective to bind to VEGFR2 ECD.

FIGS. 16A-B. Structures and binding isotherms of GU40C and itsderivatives. (FIG. 16A) Structures of GU40C(2) and GU40C(3). R: pleasesee FIGS. 15A-B. (FIG. 16B) Binding isotherms of GU40C and itsderivatives. Only GU40C and GU40C(2) able to display binding to VEGFR2ECD.

FIGS. 17A-B. GU40C truncation study results. (FIG. 17A) Downward arrowsshow GU40C truncation positions (FIG. 17B) Competitive binding assayresults; increasing concentrations of unlabelled GU40C and truncatedderivatives were competed with a constant amount of fluoraceinatedGU40C. Symbols represent; GU40C (□), 8-mer (▴), 7-mer (▾), 6-mer (⋄),5-mer (∘), 4-mer (▪), control (x).

FIG. 18. Structure of the GU40C with highlighted residues that areproposed to constitute the minimal pharmacophore.

FIG. 19. Chemical structures of GU40C (top) and GU81. GU40C was one offive hits isolated originally from screening a library of ≈250,000peptoids for specific binding to human VEGFR2. GU81 is the result of amedicinal chemistry exercise aimed at improving the potency of themolecule. This derivative binds the VEGFR2 about 4 times more tightlythan the GU40C parent.

FIG. 20. The chemical structure of the GU81 dimer. This molecule has ahigh affinity for the VEGFR2 and is a potent antagonist of its activityin vitro and in vivo.

FIG. 21. Effect of doxorubicin (Dox) and the GU81 dimer (peptoid) on thegrowth of an aggressive MMTV-induced tumor in a mouse. The data showthat while the peptoid alone did not result in significant reduction inthe rate of tumor growth, it did function synergistically with Dox.

FIG. 22. The effect of Dox and the GU81 dimer on the weight of the MMTVtumor as measured at the time of sacrifice (age 60 days). In agreementwith the measurement of tumor volume (see FIG. 21), the peptoid actssynergistically with Dox to reduce the growth of the tumor mass.

FIG. 23. Schematic of the screening technology and its application tothe identification of VEGFR2 ligands.

FIG. 24. A three-color assay to identify compounds that discriminatebetween two closely related receptors.

FIG. 25. Results of the screen against two T cell populations. Oneisolated from EAE mice (labeled red) and the other from healthy controlmice (labeled green).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The inventors and others have demonstrated that libraries of peptoids(oligo-N-substituted glycines) are rich sources of protein-bindingligands (Alluri et al., 2003; Zuckermann et al., 1994). Indeed, screensof combinatorial peptoid libraries result in the isolation ofprotein-binding peptoids that exhibit affinities and specificities quitesimilar to those exhibited by protein-binding peptides isolated fromphage display libraries or other common means (Simon et al., 1992).However, peptoids are not sensitive to peptidases or proteases (Simon etal., 1992) and are even easier and more economical to synthesize thanpeptides (Zuckermann et al., 1992). Thus, peptoids would appear to beviable candidates for antagonists of receptor-hormone interactions.Based on the limitations of the antibody- and peptide-based approachesto antagonizing hormone-receptor interactions, the inventors decided toexplore an alternative approach.

Using VEGFR2 as a model system, the inventors developed a novelcell-based binding screen that allows very large libraries of peptoidsdisplayed on beads to be screened for receptor-binding compounds. Thisassay does not require elaborate automated instrumentation as is thecase for functional screens of receptor activity. The inventorsdemonstrated that the peptoids isolated from such a screen bind VEGFR2with dissociation constants in the low μM region, an affinity comparableto that of receptor-binding peptides, but much weaker than thatexhibited by monoclonal antibodies (Witte et al., 1998). However, asimple dimerization strategy was employed to create low nM VEGFR2 leadcompounds, an affinity that is comparable with that exhibited by anantibody. The inventors further showed that one of these dimericpeptoids is capable of acting as an antagonist of VEGF-VEGFR2 binding,and thus blocks hormone-dependent receptor activation and angiogenesisin vitro. This technology constitutes a general approach to theisolation of relatively low molecular mass, high affinity, serum-stableantagonists of receptor-hormone interactions. These, and other aspectsof the invention, are described in great detail in the following pages.

1. Peptoids and Peptoid Array

The inventors synthesized a library of peptoids with a theoreticaldiversity of approximately 250,000 compounds on TentaGel beads. Thedesign of the library (FIG. 1B) attempted to take some advantage of thestructure of the VEGF-VEGR2 complex. The receptor consists of sevenextracellular immunoglobulin-like domains, a single transmembraneregion, and an intracellular tyrosine kinase domain (Matthews et al.,1991; Terman et al., 1991). VEGF binds to extracellular domains 2 and 3(Fuh et al., 1998) of VEGFR2. It has been reported that Isoleucine(Ile-43, 46, 83), Glutamate (Glu-64), Phenylalanine (Phe-17), Glutamine(Gln-79), Lysine (Lys-84) and Proline (Pro-85) side chains of VEGF arethe most important moieties for the binding to VEGFR2 (Muller et al.,1997). Therefore, in designing this peptoid library, the inventorsdecided to have at least two of the above residues fixed with the intentof biasing the library for VEGFR2 binding. The inventors selectedLys-like and Leu-like residues to be fixed at the C-terminus of eachmolecule in the library. Leu was chosen instead of Ile because Ile wasnot validated for peptoid synthesis at the time of synthesis, and Leuwas the most suitable substitute. Also, the inventors decided to placeone additional Lys-like residue in between the resin and the 8-merpeptoid, making the full-length of each peptoid a total of nineresidues. The positively-charged Lys-like residue can repel the peptoidsfrom each other on the bead surface and would avoid aggregation of thepeptoids that could hinder proper display. The library was synthesizedon TentaGel macrobeads (140-170 μM diameter), which have excellentstability and swelling properties and also provide a non-sticky surfacethat is ideal for reducing non-specific binding in screening experiments(Alluri et al., 2003). Synthesis of the library was conducted usingeight different amines (FIG. 1B) resulting in a theoretical diversity of262,144 compounds.

A. Peptoid Array Synthesis

9-mer peptoid library was synthesized on TentaGel macrobeads (140-170μm; substitution: 0.48 mmol/g resin; Rapp Polymere) using microwave(1000 W) assisted synthesis protocol. TentaGel macrobeads weredistributed equally into eight peptide synthesis reaction vessels,swelled in dimethylformamide (DMF) and each reaction vessel was treatedwith 2M Bromoacetic acid and 3.2M Di-isopropylcarbodiimide (DIC) thecoupling was performed in microwave oven set to deliver 10% power (2×15sec). After washing the beads with DMF, each vessel was treated with oneof the eight primary amines at 2M concentration and again the couplingwas performed in microwave as described above. Beads were washed,pooled, randomized and were redistributed equally into eight peptidesynthesis vessels, and the procedure was repeated until the desiredlength is achieved. For first three fixed residues (Nleu-Nlys-Nlys),each step was followed as above except the pooling step. At thecompletion of the library synthesis, beads were treated with 95% TFA,2.5% tri-isopropylsilane and 2.5% water for 2 h to remove side chainprotection groups and were neutralized with 10% di-isopropylethylaminein DMF. Finally, washed with dichloromethane, dried and stored at 4° C.(Alluri et al., 2003).

B. Synthesis of GU40C, GU40C4, GU81 and GU81 Dimer

In both monomeric (GU40C) and dimeric (GU40C4) forms, resynthesis ofpeptoid ligands were conducted on Knorr Amide MBHA resin. After loadingCys residue, general microwave assisted protocol was used to build thepeptoid portion and finally Fluorescein-5-maleimide and MaleimidePEO2-Biotin (Pierce) were coupled.

C. Linkers

The present invention may comprise multimeric species—dimers, trimers orother multimers of monomeric peptoids—that can be synthesized bysolid-phase methods, for example by first attaching a lysine or otherdiamine-containing compound to the bead, followed by growing the peptoidchains from each amino group, or created first and then joined via alinker. Any of a wide variety of linkers may be utilized to effect thejoinder of peptoids. Certain linkers will generally be preferred overother linkers, based on differing pharmacologic characteristics andcapabilities. In particular, the linkers will be attached at the free—OH group of GU40C, and may be represented by an R group substituted forthat —OH group, i.e., —C(═O)—R—C(═O)—.

Cross-linking reagents are used to form molecular bridges that tietogether functional groups of two molecules. Linking/coupling agentsused to combine to peptoids of the present invention include linkagessuch as avidin-biotin, amides, esters, thioesters, ethers, thioethers,phosphoesters, phosphoramides, anhydrides, disulfides, and ionic andhydrophobic interactions.

TABLE 1 HETERO-BIFUNCTIONAL CROSS-LINKERS Linker Reactive TowardAdvantages and Applications Spacer Arm Length SMPT Primary aminesGreater stability 11.2 A Sulfhydryls SPDP Primary amines Thiolation 6.8A Sulfhydryls Cleavable cross-linking LC-SPDP Primary amines Extendedspacer arm 15.6 A Sulfhydryls Sulfo-LC-SPDP Primary amines Extendedspacer arm 15.6 A Sulfhydryls Water-soluble SMCC Primary amines Stablemaleimide reactive group 11.6 A Sulfhydryls Enzyme-antibody conjugationHapten-carrier protein conjugation Sulfo-SMCC Primary amines Stablemaleimide reactive group 11.6 A Sulfhydryls Water-solubleEnzyme-antibody conjugation MBS Primary amines Enzyme-antibodyconjugation 9.9 A Sulfhydryls Hapten-carrier protein conjugationSulfo-MBS Primary amines Water-soluble 9.9 A Sulfhydryls SIAB Primaryamines Enzyme-antibody conjugation 10.6 A Sulfhydryls Sulfo-SIAB Primaryamines Water-soluble 10.6 A Sulfhydryls SMPB Primary amines Extendedspacer arm 14.5 A Sulfhydryls Enzyme-antibody conjugation Sulfo-SMPBPrimary amines Extended spacer arm 14.5 A Sulfhydryls Water-solubleEDC/Sulfo-NHS Primary amines Hapten-Carrier conjugation 0 Carboxylgroups ABH Carbohydrates Reacts with sugar groups 11.9 A Nonselective

An exemplary hetero-bifunctional cross-linker contains two reactivegroups: one reacting with primary amine group (e.g., N-hydroxysuccinimide) and the other reacting with a thiol group (e.g., pyridyldisulfide, maleimides, halogens, etc.). Through the primary aminereactive group, the cross-linker may react with the lysine residue(s) ofone protein (e.g., the selected antibody or fragment) and through thethiol reactive group, the cross-linker, already tied up to the firstprotein, reacts with the cysteine residue (free sulfhydryl group) of theother protein (e.g., the selective agent).

It is particular that a cross-linker having reasonable stability inblood will be employed. Numerous types of disulfide-bond containinglinkers are known that can be successfully employed to conjugatetargeting and therapeutic/preventative agents. Linkers that contain adisulfide bond that is sterically hindered may prove to give greaterstability in vivo, preventing release of the targeting peptide prior toreaching the site of action. These linkers are thus one group of linkingagents.

Another cross-linking reagent is SMPT, which is a bifunctionalcross-linker containing a disulfide bond that is “sterically hindered”by an adjacent benzene ring and methyl groups. It is believed thatsteric hindrance of the disulfide bond serves a function of protectingthe bond from attack by thiolate anions such as glutathione which can bepresent in tissues and blood, and thereby help in preventing decouplingof the conjugate prior to the delivery of the attached agent to thetarget site.

The SMPT cross-linking reagent, as with many other known cross-linkingreagents, lends the ability to cross-link functional groups such as theSH of cysteine or primary amines (e.g., the epsilon amino group oflysine). Another possible type of cross-linker includes thehetero-bifunctional photoreactive phenylazides containing a cleavabledisulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido)ethyl-1,3′-dithiopropionate. The N-hydroxy-succinimidyl group reactswith primary amino groups and the phenylazide (upon photolysis) reactsnon-selectively with any amino acid residue.

In addition to hindered cross-linkers, non-hindered linkers also can beemployed in accordance herewith. Other useful cross-linkers, notconsidered to contain or generate a protected disulfide, include SATA,SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1988). The use of suchcross-linkers is well understood in the art. Another embodiment involvesthe use of flexible linkers.

U.S. Pat. No. 4,680,338, describes bifunctional linkers useful forproducing conjugates of ligands with amine-containing polymers and/orproteins, especially for forming antibody conjugates with chelators,drugs, enzymes, detectable labels and the like. U.S. Pat. Nos. 5,141,648and 5,563,250 disclose cleavable conjugates containing a labile bondthat is cleavable under a variety of mild conditions. This linker isparticularly useful in that the agent of interest may be bonded directlyto the linker, with cleavage resulting in release of the active agent.Preferred uses include adding a free amino or free sulfhydryl group to aprotein, such as an antibody, or a drug.

U.S. Pat. No. 5,856,456 provides peptide linkers for use in connectingpolypeptide constituents to make fusion proteins, e.g., single-chainantibodies. The linker is up to about 50 amino acids in length, containsat least one occurrence of a charged amino acid (preferably arginine orlysine) followed by a proline, and is characterized by greater stabilityand reduced aggregation. U.S. Pat. No. 5,880,270 disclosesaminooxy-containing linkers useful in a variety of immunodiagnostic andseparative techniques.

Peptide linkers that include a cleavage site for an enzymepreferentially located or active within a tumor environment also arecontemplated. Exemplary forms of such peptide linkers are those that arecleaved by urokinase, plasmin, thrombin, Factor Ixa, Factor Xa, or ametallaproteinase, such as collagenase, gelatinase, or stromelysin.

D. Variants or Analogs of GU40C and GU40C4

Generic Formula. As discussed below in the Examples, hundreds ofderivatives of the parent GU40C peptoid were made in which modestalterations in the three critical side chains were introduced in aneffort to improve the “fit” of this region of the peptoid with thereceptor. One of these was the GU81 peptoid, described above, which gavea 4-fold better binding to the receptor than did the parent GU40Cmolecule. Also as discussed below, the dimer of GU81 is active in an invivo assay in combination with doxorubicin against in particularlyaggressive mouse cancer model. The formula below shows variant andinvariant locations by position number:

In addition to constraints on the side chains, as indicated above, thecarbonyl groups shown in the yellow boxes have also been found to beimportant for binding.

Substitutional Variants. It also is contemplated in the presentinvention that variants or analogs of GU40C and GU40C4 peptoids may alsoinhibit VEGF signaling through VEGFR2. Sequence variants of GU40C andGU40C4, primarily making conservative substitutions, may provideimproved compositions. Substitutional variants typically contain theexchange of one amino acid or amino acid analog for another at one ormore sites within the molecule, and may be designed to modulate one ormore properties of the molecule, in particular the affinity of themolecule for the target, without the loss of other functions orproperties.

Altered Amino Acids. As shown above, peptoids may employ modified,non-natural and/or unusual amino acids. A table of exemplary, but notlimiting, modified, non-natural and/or unusual amino acids is providedherein below. Chemical synthesis may be employed to incorporate suchamino acids into the peptides of interest.

TABLE 2 Modified, Non-Natural and Unusual Amino Acids Abbr. Amino AcidAbbr. Amino Acid Aad 2-Aminoadipic acid EtAsn N-Ethylasparagine Baad3-Aminoadipic acid Hyl Hydroxylysine Bala beta-alanine, beta-Amino- Ahylallo-Hydroxylysine propionic acid Abu 2-Aminobutyric acid 3Hyp3-Hydroxyproline 4Abu 4-Aminobutyric acid, 4Hyp 4-Hydroxyprolinepiperidinic acid Acp 6-Aminocaproic acid Ide Isodesmosine Ahe2-Aminoheptanoic acid Aile allo-Isoleucine Aib 2-Aminoisobutyric acidMeGly N-Methylglycine, sarcosine Baib 3-Aminoisobutyric acid MeIleN-Methylisoleucine Apm 2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu2,4-Diaminobutyric acid MeVal N-Methylvaline Des Desmosine Nva NorvalineDpm 2,2′-Diaminopimelic acid Nle Norleucine Dpr 2,3-Diaminopropionicacid Orn Ornithine EtGly N-Ethylglycine

Mimetics. In addition to the variants discussed above, the presentinventors also contemplate that structurally similar compounds may beformulated to mimic the key portions of peptoids of the presentinvention. Such compounds, which may be termed peptidomimetics, may beused in the same manner as the peptides of the invention and, hence,also are functional equivalents.

Certain mimetics that mimic elements of protein secondary and tertiarystructure are described in Johnson et al. (1993). The underlyingrationale behind the use of peptide mimetics is that the peptidebackbone of proteins exists chiefly to orient amino acid side chains insuch a way as to facilitate molecular interactions, such as those ofantibody and/or antigen. A peptide mimetic is thus designed to permitmolecular interactions similar to the natural molecule.

Some successful applications of the peptide mimetic concept have focusedon mimetics of β-turns within proteins, which are known to be highlyantigenic. Likely β-turn structure within a polypeptide can be predictedby computer-based algorithms, as discussed herein. Once the componentamino acids of the turn are determined, mimetics can be constructed toachieve a similar spatial orientation of the essential elements of theamino acid side chains.

Other approaches have focused on the use of small,multidisulfide-containing proteins as attractive structural templatesfor producing biologically active conformations that mimic the bindingsites of large proteins (Vita et al., 1998). A structural motif thatappears to be evolutionarily conserved in certain toxins is small (30-40amino acids), stable, and high permissive for mutation. This motif iscomposed of a β sheet and an α-helix bridged in the interior core bythree disulfides.

β-II turns have been mimicked successfully using cyclic L-pentapeptidesand those with D-amino acids (Weisshoff et al., 1999). Also, Johannessonet al. (1999) report on bicyclic tripeptides with reverse turn inducingproperties.

Methods for generating specific structures have been disclosed in theart. For example, α-helix mimetics are disclosed in U.S. Pat. Nos.5,446,128; 5,710,245; 5,840,833; and 5,859,184. Theses structures renderthe peptide or protein more thermally stable, also increase resistanceto proteolytic degradation. Six, seven, eleven, twelve, thirteen andfourteen membered ring structures are disclosed.

Methods for generating conformationally restricted beta turns and betabulges are described, for example, in U.S. Pat. Nos. 5,440,013;5,618,914; and 5,670,155. Beta-turns permit changed side substituentswithout having changes in corresponding backbone conformation, and haveappropriate termini for incorporation into peptides by standardsynthesis procedures. Other types of mimetic turns include reverse andgamma turns. Reverse turn mimetics are disclosed in U.S. Pat. Nos.5,475,085 and 5,929,237, and gamma turn mimetics are described in U.S.Pat. Nos. 5,672,681 and 5,674,976.

E. Purification of Peptoids

It may be desirable to purify peptoids according to the presentinvention. Purification techniques are well known to those of skill inthe art. These techniques typically involve chromatographic andelectrophoretic techniques to achieve partial or complete purification(or purification to homogeneity). Analytical methods particularly suitedto the preparation of a pure peptoid are ion-exchange chromatography,exclusion chromatography; polyacrylamide gel electrophoresis;isoelectric focusing. A particularly efficient method of purifyingpeptoids is fast protein liquid chromatography or even HPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of a peptoid.The term “purified peptoid” as used herein, is intended to refer to acomposition, isolatable from other components, wherein the peptoid ispurified to any degree relative to its normally-obtainable state. Apurified peptoid therefore also refers to a peptoid free from theenvironment in which it may normally occur.

Generally, “purified” will refer to a peptoid composition that has beensubjected to fractionation to remove various other components, and whichcomposition substantially retains its expressed biological activity.Where the term “substantially purified” is used, this designation willrefer to a composition in which the peptoid forms the major component ofthe composition, such as constituting about 50%, about 60%, about 70%,about 80%, about 90%, about 95% or more of the composition by weight.

Various methods for quantifying the degree of purification of thepeptoid will be known to those of skill in the art in light of thepresent disclosure. These include, for example, determining the specificactivity of an active fraction, or assessing the amount of peptoidwithin a fraction by SDS/PAGE analysis. A preferred method for assessingthe purity of a fraction is to calculate the specific activity of thefraction, to compare it to the specific activity of the initial extract,and to thus calculate the degree of purity, herein assessed by a “-foldpurification number.” The actual units used to represent the amount ofactivity will, of course, be dependent upon the particular assaytechnique chosen to follow the purification and whether or not thepeptoid exhibits a detectable activity.

Various techniques suitable for use in peptoid purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified peptoid.

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special typeof partition chromatography that is based on molecular size. The theorybehind gel chromatography is that the column, which is prepared withtiny particles of an inert substance that contain small pores, separateslarger molecules from smaller molecules as they pass through or aroundthe pores, depending on their size. As long as the material of which theparticles are made does not adsorb the molecules, the sole factordetermining rate of flow is the size. Hence, molecules are eluted fromthe column in decreasing size, so long as the shape is relativelyconstant. Gel chromatography is unsurpassed for separating molecules ofdifferent size because separation is independent of all other factorssuch as pH, ionic strength, temperature, etc. There also is virtually noadsorption, less zone spreading and the elution volume is related in asimple matter to molecular weight.

Affinity Chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculethat it can specifically bind to. This is a receptor-ligand typeinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (alter pH, ionic strength, temperature, etc.).

A particular type of affinity chromatography useful in the purificationof carbohydrate containing compounds is lectin affinity chromatography.Lectins are a class of substances that bind to a variety ofpolysaccharides and glycoproteins. Lectins are usually coupled toagarose by cyanogen bromide. Conconavalin A coupled to Sepharose was thefirst material of this sort to be used and has been widely used in theisolation of polysaccharides and glycoproteins other lectins that havebeen include lentil lectin, wheat germ agglutinin which has been usefulin the purification of N-acetyl glucosaminyl residues and Helix pomatialectin. Lectins themselves are purified using affinity chromatographywith carbohydrate ligands. Lactose has been used to purify lectins fromcastor bean and peanuts; maltose has been useful in extracting lectinsfrom lentils and jack bean; N-acetyl-D galactosamine is used forpurifying lectins from soybean; N-acetyl glucosaminyl binds to lectinsfrom wheat germ; D-galactosamine has been used in obtaining lectins fromclams and L-fucose will bind to lectins from lotus.

The matrix should be a substance that itself does not adsorb moleculesto any significant extent and that has a broad range of chemical,physical and thermal stability. The ligand should be coupled in such away as to not affect its binding properties. The ligand should alsoprovide relatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand. One of the mostcommon forms of affinity chromatography is immunoaffinitychromatography. The generation of antibodies that would be suitable foruse in accord with the present invention is discussed below.

2. VEGF

A. VEGF

Vascular endothelial growth factor (VEGF) is an important signalingprotein involved in both vasculogenesis (the de novo formation of theembryonic circulatory system) and angiogenesis (the growth of bloodvessels from pre-existing vasculature). As its name implies, VEGFactivity has been mostly studied on cells of the vascular endothelium,although it does have effects on a number of other cell types (e.g.,stimulation monocyte/macrophage migration, neurons, cancer cells, kidneyepithelial cells). In vitro, VEGF has been shown to stimulateendothelial cell mitogenesis and cell migration. VEGF is also avasodilator and increases microvascular permeability and was originallyreferred to as vascular permeability factor.

The broad term “VEGF” covers a number of proteins from two families,that result from alternate splicing of mRNA from a single, 8 exon, VEGFgene. The two different families are referred to according to theirterminal exon (exon 8) splice site—the proximal splice site (denotedVEGF_(xx)) or distal splice site (VEGF_(xxb)). In addition, alternatesplicing of exon 6 and 7 alters their heparin binding affinity, andamino acid number (in humans: VEGF₁₂₁, VEGF₁₂₁b, VEGF₁₄₅, VEGF₁₆₅,VEGF₁₆₅b, VEGF₁₈₉, VEGF₂₀₆; the rodent orthologs of these proteinscontain one fewer amino acid). These domains have important functionalconsequences for the VEGF splice variants as the terminal (exon 8)splice site determines whether the proteins are pro-angiogenic (proximalsplice site, expressed during angiogenesis) or anti-angiogenic (distalsplice site, expressed in normal tissues). In addition, inclusion orexclusion of exons 6 and 7 mediate interactions with heparan sulfateproteoglycans (HSPGs) and neuropilin co-receptors on the cell surface,enhancing their ability to bind and activate the VEGF signalingreceptors (VEGFR's).

The VEGF splice variants are released from cells as glycosylateddisulfide-bonded dimers. Structurally VEGF belongs to the PDGF family ofcystine-knot growth factors. Subsequently, several closely-relatedproteins were discovered (Placenta growth factor (PlGF), VEGF-B, VEGF-Cand VEGF-D) which together comprise the VEGF sub-family of growthfactors. VEGF is sometimes referred to as VEGF-A to differentiate itfrom these related growth factors. A number of VEGF-related proteinshave also been discovered encoded by viruses (VEGF-E) and in the venomof some snakes (VEGF-F).

B. VEGFR

All members of the VEGF family stimulate cellular responses by bindingto tyrosine kinase receptors (the VEGFRs) on the cell surface, causingthem to dimerize and become activated through transphosphorylation,although to different sites, times and extents. The VEGF receptors havean extracellular portion consisting of 7 immunoglobulin-like domains, asingle transmembrane spanning region and an intracellular portioncontaining a split tyrosine-kinase domain. VEGF-A binds to VEGFR-1(Flt-1) and VEGFR-2 (KDR/Flk-1). VEGFR-2 appears to mediate almost allof the known cellular responses to VEGF. The function of VEGFR-1 is lesswell defined, although it is thought to modulate VEGFR-2 signaling.Another function of VEGFR-1 may be to act as a dummy/decoy receptor,sequestering VEGF from VEGFR-2 binding (this appears to be particularlyimportant during vasculogenesis in the embryo). VEGF-C and VEGF-D, butnot VEGF-A, are ligands for a third receptor (VEGFR-3), which mediateslymphangiogenesis.

C. Known Antagonists of VEGFR

Anti-VEGF therapies are important advances in the treatment of certaincancers. They can be monoclonals such as Bevacizumab (Avastin®), or oralsmall molecules that inhibit the tyrosine kinases stimulated by VEGF,such as Sunitinib, Sorafenib, Axitinib, Pazopanib.

3. Screening Assays

A. Peptoid Variants

The present invention also contemplates the screening of peptoidvariants of GU40C4 for their ability to bind to VEGFR2 and to inhibitangiogenesis. In addition to performing the binding assay described inthe Examples, by which GU40C4 was identified, various other assays mayalso be conducted, such as in vitro and in vivo binding and inhibitionassays, as well as assays for particular therapeutic efficacy, e.g.,anti-cancer, anti-macular degeneration and anti-angiogenesis.

The present invention provides methods of screening for agents that bindVEGFR2. In an embodiment, the present invention is directed to a methodof:

(a) providing a candidate peptoid;

(b) contacting the peptoid with VEGFR2; and

I determining the binding of the candidate peptoid with VEGFR2, whereinbinding to VEFGR2 identifies the candidate as a putative inhibitor ofangiogenesis.

Measuring binding to may be direct, by identifying a VEGFR2-candidatecomplex, by identifying labeled candidate associated with VEGFR2, or byassessing the inhibition of binding of a labeled peptoid to VEGFR2.

It will, of course, be understood that all the screening methods of thepresent invention are useful in themselves notwithstanding the fact thateffective candidates may not be found. The invention provides methodsfor screening for such candidates, not solely methods of finding them.

Various cells that express VEGFR2 can be utilized for screening ofcandidate substances. Exemplary cells include, but are not limited toporcine aortic endothelial cells and human vascular endothelial cells(HUVECs). Depending on the assay, culture may be required. Labeledcandidate peptoids may be contacted with the cell and binding assessedtherein. Various readouts for binding of candidate substances to cellsmay be utilized, including ELISA, fluorescent microscopy and FACS. Inparticular, the assay shown in FIG. 1A may be utilized in variousformats to identify peptoids of interest.

The present invention particularly contemplates the use of variousanimal models. For example, various animal models of cancer may be usedto determine if the candidate peptoids inhibits cancer cell growth,metastasis or recurrence, or affects its ability to evade the effects ofother drugs. Treatment of these animals with test compounds will involvethe administration of the compound, in an appropriate form, to theanimal. Administration will be by any route the could be utilized forclinical or non-clinical purposes, including but not limited to oral,nasal, buccal, or even topical. Alternatively, administration may be byoral, sublingual, intratracheal instillation, bronchial instillation,intradermal, subcutaneous, intramuscular, intraperitoneal or intravenousinjection. Specifically contemplated are intratumoral and intraocularadministration and regional (to a tumor or eye) administration.

B. Cell Based Screening Formats

Another aspect of the invention involves cell based screening assaysthat identify target-specific ligands, such as peptoids. Cells havingdifferential characteristics, such as the presence or absence of a cellsurface receptor, but otherwise identical, are differentially labeled(e.g., two different colored quantum dots). The cells are then mixed inan approximately 1:1 ratio and then exposed to a library of moleculesdisplayed on hydrophilic beads. After appropriate incubation andwashing, the beads that bind only one color cell are picked. The beadsare treated to remove the cells and other debris, and the bound moleculeis identified by an appropriate analytical technique. This two-colorassay demands extremely high specificity. If the bead-displayed moleculebinds any other molecule on the cell surface other than the targetreceptor, then both colored cells will be retained and the molecule willnot be identified as a hit. See Udugamasooriya et al. (2008).

The assay can be modified to accommodate a variety of different formats.For example, a three cell types assay can be used to distinguish ligandsthat bind to highly related molecules. For example, where two receptorsare almost identical, cells are provided that are null or have one orthe other related receptor. Each cell type (null, receptor 1-containingand receptor 2-containing) is labeled with a different agent (e.g.,colored quantum dot). The cells are mixed together in an approximately1:1:1 ratio and exposed to a bead library. Beads that bind only onecolor cell are picked and the chemical that they display ischaracterized (see FIG. 24).

Examples of structures that can be differentiated include antibody orT-cell receptors of various immune cells, growth factor receptors, cellmatrix proteins, lectins, carbohydrates, lipids, cell surface antigensfrom various pathogens. Additionally, the cells could differ not in thecomposition of the cell surface molecules, but in their arrangement. Forexample on one cell type, two given cell surface molecules mightassociate with one another and provide a unique binding site for aligand that might be absent from a different cell type where thesereceptors do not associate. Labeling can utilize calorimetric,fluorimetric, bioluminescent or chemilluminescent labels.

The assay can also be modified to identify ligands that bind to cellspresent in only one of two or more distinct cell populations. Forexample, all CD4+ T cells from a healthy individual or group ofindividuals could be labeled with one colored dye and the CD4+ T cellsfrom an individual or group of individuals with an autoimmune diseasecould be labeled with a different colored dye. The two populations of Tcells could then be mixed with the bead library and beads retaining onlycells from the autoimmune patients could be selected. These T cellswould be candidates for the autoimmune T cells that display the T cellReceptor (TCR) that binds the autoantigen and contributes to disease,since these cells should only be abundant in the autoimmune samples andnot in cells obtained from healthy individuals.

In another application, the two or more cell populations could differsolely in the presence or absence of a genetic mutation that mightresult in a change in the composition and/or organization of moleculeson the cell surface. An example would be cells that contain wild-type ormutant ras gene (e.g., K-ras).

4. Treating Cancers

In one aspect of the present invention, one may utilize the peptoidcompounds of the present invention and analogs thereof in the inhibitionof cancer. Any of a variety of different cancers, particularly solidtumors, are contemplated as suitable for treatment with the presentinvention. For example, a glioma cell, a sarcoma cell, a myeloma cellmay be treated with peptoids, as can cancer cells such as a lung cancercell, a skin cancer cell, a head & neck cancer cell, a stomach cancercell, a breast cancer cell, a colon cancer cell, a pancreatic cancercell, a liver cancer, an ovarian cancer cell, a uterine cancer cell, acervical cancer cell, a testicular cancer cell, a rectal cancer cell, anesophageal cancer cell, or a brain cancer cell. The cancer may also beprimary, metastatic, multi-drug resistant or recurrent.

A. Monotherapy

In one embodiment, the subject will be administered peptoid or variants,mimetics or analogs thereof. Formulations would be selected based on theroute of administration and purpose including, but not limited to,parenteral formulations, topical formulations, liposomal formulationsand classic pharmaceutical preparations for oral administration.Particular routes include intratumoral injection and injection into thetumor cell vasculature. Repeated or continuous therapy over a period oftime (weeks to months) also is contemplated.

B. Combined Therapy

In order to increase the effectiveness of peptoid therapy, it may bedesirable to combine these compositions with another agent effective inthe treatment of cancer. The terms “contacted” and “exposed,” whenapplied to a cell, tissue or organism, are used herein to describe theprocess by which an peptoid therapy and/or other agent are delivered toa target cell, tissue or organism or are placed in direct juxtapositionwith the target cell, tissue or organism. Other anti-cancer agentsinclude, but are not limited to.

The peptoid treatment may precede, be concurrent with and/or follow theother agent(s) by intervals ranging from minutes to weeks. Inembodiments where the peptoid treatment and other agent(s) are appliedseparately to a cell, tissue or organism, one would generally ensurethat a significant period of time did not expire between the time ofeach delivery, such that the peptide and agent(s) would still be able toexert an advantageously combined effect on the cell, tissue or organism.For example, in such instances, it is contemplated that one may contactthe cell, tissue or organism with two, three, four or more modalitiessubstantially simultaneously (i.e., within less than about a minute)with the peptoid or mimic or analog. In other aspects, one or moreagents may be administered within of from substantially simultaneously,about 1 minute, about 5 minutes, about 10 minutes, about 20 minutesabout 30 minutes, about 45 minutes, about 60 minutes, about 2 hours,about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7hours about 8 hours, about 9 hours, about 10 hours, about 11 hours,about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48hours, about 3 days, about 4 days, about 5 days, about 6 days, about 7days, about 8 days, about 9 days, about 10 days, about 11 days, about 12days, about 13 days, about 14 days, about 21 days, about 4 weeks, about5 weeks, about 6 weeks, about 7 week or about 8 weeks or more, and anyrange derivable therein, prior to and/or after administering peptoid ormimic or analog thereof.

For example, as shown in FIG. 26, treatment of a mouse bearing a tumorwith the GU81 dimer and the classical anti-cancer compound doxorubicinis more effective in slowing tumor growth than either compound alone.

Various combination regimens of the peptoid treatment and one or moreother anti-pain agents may be employed. Non-limiting examples of suchcombinations are shown below, wherein a peptoid composition is “A” andthe other anti-cancer agent is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Other combinations are contemplated. Again, to achieve cancer cellinhibition, both agents are delivered to a cell in a combined amounteffective to achieve the desired inhibition, which may include cellstasis or cell death.

Administration of the peptoid composition to a cell, tissue or organismmay follow general protocols for the administration of pharmaceuticals,taking into account the toxicity, if any. It is expected that thetreatment cycles would be repeated as necessary. In particularembodiments, it is contemplated that various additional agents may beapplied in any combination with the present invention.

Agents or factors suitable for use in a combined therapy are anychemical compound or treatment method that may also produce anadvantageous effect, alone or in combination with GU40C4. Such agentsand factors include radiation and waves that induce DNA damage such as,γ-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions,and the like. A variety of chemical compounds, also described as“chemotherapeutic agents,” function to induce DNA damage, all of whichare intended to be of use in the combined treatment methods disclosedherein. Chemotherapeutic agents contemplated to be of use, include,e.g., doxorubicin, verapamil, podophyllotoxin, adriamycin,5-fluorouracil (5FU), etoposide (VP-16), camptothecin, actinomycin-D,mitomycin C, cisplatin (CDDP) and even hydrogen peroxide.

C. Pharmaceutical Formulations

Pharmaceutical formulations of the present invention comprise aneffective amount of a peptoid dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refer to compositions that do not producean adverse, allergic or other untoward reaction when administered to ananimal, such as, for example, a human, as appropriate. The preparationof such pharmaceutical compositions are known to those of skill in theart in light of the present disclosure, as exemplified by Remington'sPharmaceutical Sciences, 18^(th) Ed. Mack Printing Company, 1990,incorporated herein by reference. Moreover, for animal (e.g., human)administration, it will be understood that preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art. Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the therapeutic orpharmaceutical compositions is contemplated.

The pharmaceuticals of the present invention may comprise differenttypes of carriers depending on whether it is to be administered insolid, liquid or aerosol form, and whether it need to be sterile forsuch routes of administration as injection. The present invention can beadministered intravenously, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostaticaly, intrapleurally, intratracheally, intranasally,intravitreally, intravaginally, intrarectally, topically,intratumorally, intramuscularly, intraperitoneally, subcutaneously,subconjunctival, intravesicularlly, mucosally, intrapericardially,intraumbilically, intraocularally, orally, topically, locally,inhalation (e.g., aerosol), injection, infusion, continuous infusion,localized perfusion bathing target cells directly, via a catheter, via alavage, in cremes, in lipid compositions (e.g., liposomes), or by othermethod or any combination of the forgoing as would be known to one ofordinary skill in the art.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 50 microgram/kg/body weight, about 100 microgram/kg/bodyweight, about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1 milligram/kg/bodyweight, about 5 milligram/kg/body weight, about 10 milligram/kg/bodyweight, about 50 milligram/kg/body weight, about 100 milligram/kg/bodyweight, about 200 milligram/kg/body weight, about 350 milligram/kg/bodyweight, about 500 milligram/kg/body weight, to about 1000 mg/kg/bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100 mg/kg/bodyweight, about 5 microgram/kg/body weight to about 500 milligram/kg/bodyweight, etc., can be administered, based on the numbers described above.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof. Thepharmaceuticals may be formulated into a composition in a free base,neutral or salt form. Pharmaceutically acceptable salts, include theacid addition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

In certain embodiments, the compositions are prepared for administrationby such routes as oral ingestion. In these embodiments, the solidcomposition may comprise, for example, solutions, suspensions,emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatincapsules), sustained release formulations, buccal compositions, troches,elixirs, suspensions, syrups, wafers, or combinations thereof. Oralcompositions may be incorporated directly with the food of the diet.Preferred carriers for oral administration comprise inert diluents,assimilable edible carriers or combinations thereof. In other aspects ofthe invention, the oral composition may be prepared as a syrup orelixir. A syrup or elixir, and may comprise, for example, at least oneactive agent, a sweetening agent, a preservative, a flavoring agent, adye, a preservative, or combinations thereof.

In certain preferred embodiments an oral composition may comprise one ormore binders, excipients, disintegration agents, lubricants, flavoringagents, and combinations thereof. In certain embodiments, a compositionmay comprise one or more of the following: a binder, such as, forexample, gum tragacanth, acacia, cornstarch, gelatin or combinationsthereof, an excipient, such as, for example, dicalcium phosphate,mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate or combinations thereof, a disintegratingagent, such as, for example, corn starch, potato starch, alginic acid orcombinations thereof, a lubricant, such as, for example, magnesiumstearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof, a flavoring agent, such as, forexample peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations thereof the foregoing. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, carriers such as a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

5. Treatment of Macular Degeneration

A. Background

Macular degeneration is a medical condition predominantly found inelderly adults in which the center of the inner lining of the eye, knownas the macula area of the retina, suffers thinning, atrophy, and in somecases bleeding. This can result in loss of central vision, which entailsinability to see fine details, to read, or to recognize faces. Accordingto the American Academy of Opthalmology, it is the leading cause ofcentral vision loss (blindness) and in the United States today for thoseover the age of fifty years. Although some macular dystrophies thataffect younger individuals are sometimes referred to as maculardegeneration, the term generally refers to age-related maculardegeneration (AMD or ARMD).

Age-related macular degeneration begins with characteristic yellowdeposits in the macula (central area of the retina) called drusen. Mostpeople with these early changes have good vision. People with drusen cango on to develop advanced AMD. The risk is considerably higher when thedrusen are large and numerous and associated with disturbance in thepigmented cell layer under the macula. Recent research suggests thatlarge and soft drusen are related to elevated cholesterol deposits andmay respond to cholesterol lowering agents or the Rheo Procedure.

Advanced AMD, which is responsible for profound vision loss, has twoforms: dry and wet. Central geographic atrophy, the dry form of advancedAMD, results from atrophy to the retinal pigment epithelial layer belowthe retina, which causes vision loss through loss of photoreceptors(rods and cones) in the central part of the eye. While no treatment isavailable for this condition, vitamin supplements with high doses ofantioxidants, Lutein and Zeaxanthin, have been demonstrated by theNational Eye Institute and others to slow the progression of dry maculardegeneration and in some patients, improve visual acuity.

Neovascular or exudative AMD, the wet form of advanced AMD, causesvision loss due to abnormal blood vessel growth in thechoriocapillaries, through Bruch's membrane, ultimately leading to bloodand protein leakage below the macula. Bleeding, leaking, and scarringfrom these blood vessels eventually cause irreversible damage to thephotoreceptors and rapid vision loss if left untreated.

Fluorescein angiography allows for the identification and localizationof abnormal vascular processes. Optical coherence tomography is now usedby most ophthalmologists in the diagnosis and the followup evaluation ofthe response to treatment by using either Avastin or Lucentis which areinjected into the vitreous of the eye at various intervals.

Until recently, no effective treatments were known for wet maculardegeneration. However, anti-VEGF (anti-Vascular Endothelial GrowthFactor) agents, when injected directly into the vitreous humor of theeye using a small, painless needle, can cause contraction of theabnormal blood vessels and improvement of vision. The injectionsfrequently have to be repeated on a monthly or bi-monthly basis.Examples of these agents include Lucentis, Avastin and Macugen. OnlyLucentis and Macugen are FDA approved as of April 2007, and onlyLucentis and Avastin appear to be able to improve vision, but theimprovements are slight and do not restore full vision. It isanticipated by GU40C4 would be used similar to how Avastin isadministered.

The Age-Related Eye Disease Study showed that a combination of high-dosebeta-carotene, vitamin C, vitamin E, and zinc can reduce the risk ofdeveloping advanced AMD by about 25% in those patients who have earlierbut significant forms of the disease. This is the only provenintervention to decrease the risk of advanced AMD at this time. A followup study, Age-Related Eye Disease Study 2 to study the potentialbenefits of lutein, zeaxanthine, and fish oil, is currently underway.

Anecortave acetate, (Retanne), is an anti-angiogenic drug that is givenas an injection behind the eye (avoiding an injection directly into theeye) that is currently being studied as a potential way of reducing therisk of neovascular (or wet) AMD in high-risk patients. Recent studiessuggest that statins, a family of drugs used for reducing cholesterollevels, may be effective in prevention of AMD, and in slowing itsprogression.

B. Treatment

In accordance with the present invention, peptoids will be utilized muchin the same way as Avastin. It is contemplated that in addition toinjection, peptoids of the present invention, because of their smallersize, may be administered in a topical solution, i.e., eye drops.Treatment can be prophylatic, prior to the development of symptoms, ormay be therapeutic in that a diagnosis and/or symptoms of AMD arepresent. Administration may be chronic, i.e., on a daily or weeklybasis.

Opthalmic formulation delivers the drug on the eye, into the eye, oronto the conjunctiva. Transcorneal transport (i.e., drug penetrationinto the eye) is not an effective process, with an estimated onlyone-tenth of a dose penetrating into the eye. Most ophthalmic solutionsare dispensed in eye dropper bottles. Patients should be shown how toproperly instill the drops in their eyes, and every effort should bemade to emphasize the need for instilling only one drop peradministration, not two or three. When more than one drop is to beadministered, wait at least five minutes between administrations.

The physiologic pH of blood and tears is approximately 7.4. Thus, from acomfort and safety standpoint, this would be the optimal pH ofophthalmic and parenteral solutions. This may not be possible, however,from a perspective of solubility, chemical stability or therapeuticactivity. Thus, some compromise must be made and product stability mustbe considered paramount. When a formulation is administered to the eye,it stimulates the flow of tears. Tear fluid is capable of quicklydiluting and buffering small volumes of added substances, thus the eyecan tolerate a fairly wide pH range. Ophthalmic solutions may range frompH 4.5-11.5, but the range to prevent corneal damage is 6.5-8.5.

Isotonic or iso-osmotic solutions do not damage tissue or produce painwhen administered are desired. Solutions which contain fewer particlesand exert a lower osmotic pressure than 0.9% saline are called hypotonicand those exerting higher osmotic pressures are referred to ashypertonic. Administration of a hypotonic solution produces painfulswelling of tissues as water passes from the administration site intothe tissues or blood cells. Hypertonic solutions produce shrinking oftissues as water is pulled from the biological cells in an attempt todilute the hypertonic solution. The eye can tolerate a range oftonicities as low as 0.6% and as high as 1.8% sodium chloride solution.Several methods are used to adjust isotonicity of pharmaceuticalsolutions.

Preservatives are commonly used in ophthalmic formulations. The FDAAdvisory Review Panel on OTC Ophthalmic Drug Products (December 1979)has established concentrations for formulations that will have directcontact with the eye.

Some drugs can be chemically degraded by oxidation, and an antioxidantshould be added to such formulations. The common agents and theirmaximum concentration used in ophthalmic formulations include sulfites(can cause allergic-type reactions in certain people) and disodiumethylenediaminetetraacetic acid.

Viscosity measures the resistance of a solution to flow when a stress isapplied. Generally the solutions are 1% or 2% and the viscosity ismeasured at 20° C. Viscosity enhancers are used in ophthalmic solutionsto increase their viscosity. This enables the formulation to remain inthe eye longer and gives more time for the drug to exert its therapeuticactivity or undergo absorption. The most common viscosity desired in anophthalmic solution is between 25 and 50 cps.

Sterility is defined as the absence of viable microbial contamination,and is an absolute requirement of all ophthalmic formulations.Therefore, ophthalmic formulations must be prepared in a laminar flowhood using aseptic techniques just the same as intravenous formulations.The sterile formulations must be packaged in sterile containers.

For additional resources, see Hecht, G. Ophthalmic Preparations, Chapter89, pp. 1563-1573; Niebergall, P. J. Ionic Solutions and ElectrolyticEquilibria, Chapter 17, pp. 225-227; Sokoloski, T. D. Solutions andPhase Equilibria, Chapter 16, pp. 206-208 and Reich, I., Schnaare, R.,Sugita, E. T. Tonicity, Osmoticity, Osmolality and Osmolarity, Chapter36, pp. 613-616, 620-627 all in Remington's, 19^(th) ed.

6. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 A. Materials and Methods

Peptoid library synthesis. 9-mer peptoid library was synthesized onTentaGel macrobeads (140-170 μm; substitution: 0.48 mM/g resin; RappPolymere) using microwave (1000 W) assisted synthesis protocol. TentaGelmacrobeads were distributed equally into eight peptide synthesisreaction vessels, swelled in dimethylformamide (DMF) and each reactionvessel was treated with 2M Bromoacetic acid and 3.2MDi-isopropylcarbodiimide (DIC) and the coupling was performed inmicrowave oven set to deliver 10% power (2×15 seconds). After washingthe beads with DMF, each vessel was treated with one of the eightprimary amines at 2M concentration and again the coupling was performedin microwave as described above. Beads were washed, pooled, randomizedand were redistributed equally into eight peptide synthesis vessels, andthe procedure was repeated until the desired length is achieved. Forfirst three fixed residues (Nleu-Nlys-Nlys), each step was followed asabove except the pooling step. At the completion of the librarysynthesis, beads were treated with 95% TFA, 2.5% tri-isopropylsilane and2.5% water for 2 h to remove side chain protection groups and wereneutralized with 10% di-isopropylethylamine in DMF. Finally, washed withdichloromethane, dried and stored at 4° C. (Alluri et al., 2003).

Bi-color on-bead cells screening assay. About 100,000 library beads wereswelled in DMF, washed with PBS and finally equilibrated in 3% BSAcontaining DMEM media for 1 h. PAE/KDR and PAE parental cells (Sibtech,Inc.) were removed from culture plates using enzyme free celldissociation buffer (Gibco), washed and re-suspended in DMEM media.Cells were labeled using quantum dots (Invitrogen) following themanufactures protocol. PAE/KDR cells labeled with Qtracker 655 (red) andPAE parental cells labeled with Qtracker 565 (green). Both labeled cellswere mixed with 1:1 ratio to give final cell density 1×10⁶ per celltype, and gently stirred to break cell clusters. Cell suspension mixturewas added to the beads containing culture plates and incubated at 37° C.with gentle shaking for 60-75 min. Beads were gently washed two timeswith DMEM media and visualized under the fluorescent microscope equippedwith the DAPI filter (10× magnification). Single positive beadscontaining fluorescently tagged red cells only were manually removedwith a 20 μl pipette using medium size pipette tips. Selected beads werewashed and boiled with 1% SDS solution for 30 min to strip off cells andsubjected to Edman sequencing in order to identify the sequence.

Dimeric library synthesis. The dimer libraries were synthesized on KnorrAmide MBHA resin (substitution: 0.78 mmol/g resin; Novabiochem). FirstFmoc-Cys(Trt)-OH was loaded onto the bead (HOBt, HBTU, DIPEA) followedby Fmoc-Lys(Dde)-OH(HOBt, DIC). Then Fmoc group was selectively removedand coupled different numbers and combinations of Fmoc-B-Ala-OH or/andFmoc-Y-Ahx-OH(HOBt, DIC) onto the N-terminal amino group of Lys inlonger dimer series. Then, both Fmoc and Dde groups were removed (2%hydrazine and 20% piperidine) and continued general microwave assistedpeptoid synthesis steps as described in the library synthesis procedureuntil the total nine residues were added. Here in each treatment, theresidues were added to both open amino ends ultimately connected by Lys(double addition). In shorter dimers, after coupling with initial Cys,Fmoc-Lys(Fmoc)-OH was coupled. Once both Fmoc groups were removedsimultaneously, started coupling peptoid residues again using themicrowave assisted protocol, which resulted in double addition as above.For truncated peptoid dimers, avoided adding C-terminal residues one ata time up to first 3 residues, which ultimately resulted shorter dimerswith 6-8 residues in each monomer. After the synthesis, peptoid dimerswere cleaved off the resin using 95% TFA, 2.5% tri-isopropylsilane and2.5% water for 2 h and purified using HPLC. Each compound was treatedwith fluorescein-5-maleimide (pH=7; Pierce) in PBS to attach FITC groupand were re-purified using HPLC.

Resynthesis of biotinylated and FITC labeled peptoids. In both monomericand dimeric forms, resynthesis of peptoid ligands were conducted onKnorr Amide MBHA resin. After loading Cys residue, general microwaveassisted protocol was used to build the peptoid portion and finallyFluorescein-5-maleimide and Maleimide PEO2-Biotin (Pierce) were coupled.

ELISA based binding assay. White, clear bottom 96-well plates (CorningInc.) were coated with of 1 μg/ml recombinant human VEGFR2 protein (R&DSystems) using the sensitizing buffer (0.621 g NaHCO₃ & 0.275 g Na₂CO₃dissolved on 100 ml of ddH₂O, pH=9.5) overnight at 4° C. Washed with3×200 μl of wash buffer 1×PBS with 0.05% Tween-20 and blocked withStartingblock blocking buffer (Pierce). Added 50 μl of serial dilutionsof FITC labeled peptoids dissolved in Startingblock blocking buffer toeach well & allowed to react for 1 h at room temperature. Washed with5×200 μl of wash buffer 1×PBS with 0.05% Tween-20 and remainingfluorescence was measured at 520 nm using the plate reader.

GU40C4 cell surface binding assay. Different types of cells (PAE/KDR,PAE, Hela, HEK-293, MCF-7, HFF) were grown on each well of chamberslides (Nalge Nunc International) (10,000 cells/chamber) overnight.Washed, and fixed cells with 3.7% formaldehyde and blocked withStartingblock blocking buffer. Each chamber was treated (except thecontrol) with 75 nm biotinylated GU40C4 peptoid followed by Qdot® 655streptavidin conjugate (Invitrogen). After the final washing, cells weremounted by ProLong® Gold antifade reagent with DAPI (Invitrogen) andvisualized under DAPI filter of the fluorescence microscope.

Western blots. Experiments were conducted using PAE/KDR cells that weregrown on 6-well plates. Overnight serum starved cells were treated with1.3 nM VEGF (Invitrogen) and different concentrations of GU40C4 peptoidor Avastin (Genentech). The blots were probed with Phospho-VEGF receptor2 (Tyr1175) (19A10) rabbit monoclonal or Total VEGFR2 primary antibodies(Cell signaling) and HRP-conjugated secondary antibody (BioRad).

HUVEC proliferation assay. HUVEC cells (ScienceCell) were harvestedusing enzyme free cell dissociation buffer and resuspended in ECM (5%FBS). Cells were plated (2000 cells/well) on white, clear bottom 96-wellplates coated with gelatin. Cells were grown for 24 h at 37° C. Changethe media which has 0.2% FBS, and treated with 1.3 nM VEGF and differentconcentrations of GU40C4 peptoid and FLAG peptide as the control. Thetreatment was repeated with fresh media (0.2% FBS) and reagents aftertwo days. After four days, viable cells were determined usingCellTiter-Glo® Luminescent Cell Viability Assay kit (Promega), and theluminescent signal was read by the plate reader.

HUVEC tube formation assay. Endothelial cell medium gel (ECM gel) wasthawed overnight at 4° C. refrigerator and added 50 μl to each well ofpre-chilled white, clear bottom 96-well plate. Incubated for 30 min tofacilitate the gel formation. HUVEC cells were harvested using enzymefree cell dissociation buffer and resuspended in ECM (0.2% FBS) andtreated with 1.3 nM VEGF and different concentrations of GU40C4 peptoidand 10 μM control peptoid, GU40C (monomer) and GU40CC (ineffectivedimer) as the controls. 150 μl of these treated cell suspension (20,000cells) was added per well and incubated at 37° C. and visualized tubeformation under the light microscope after 16 hours (10× magnification).

Cell Culture. The human Ewing's sarcoma cell line A673 (CRL-1598, ATCC,Manassas, Va.) was grown as a monolayer in Dulbeco's minimal essentialmedium (Invitrogen, Carlsbad, Calif.) supplemented with 10%heat-inactivated fetal calf serum (Atlanta Biologicals, Lawrenceville,Ga.). Cells were maintained at 37° C. in a mixture of 5% CO₂ and 95%air. Cell viability was monitored by Trypan Blue (Invitrogen, Carlsbad,Calif.) exclusion after trypsinization. Cell fingerprinting wasperformed by Dr. Luc Girard at the McDermott Center at UT Southwesternto verify identity of cells and the cells were also tested and found tobe negative for Mycoplasma prior to use.

Tumor study. Tumors were established in the right flank of 6-8 week oldfemale athymic nu/nu mice (NCl, Frederick, Md.) by subcutaneousinjection of 2.5 million cells in a volume of 50 μl PBS. On the sameday, an Alzet subcutaneous pump (DURECT Corporation, Cupertino Calif.)was implanted per manufacturer's recommendations. Briefly, the pumpswere filled with 100 μl of GU40C4 or control peptoid (8 mg/ml insaline). Pumps were weighed empty and again when filled to ensurecorrect loading. Based on the mean pumping rate (0.2 μl/hr), these pumpswere predicted to elute for 21 days. Implantation of the pump wasperformed by making a small incision on the back of the anesthetizedanimal and subsequently using a hemostat to create a subcutaneous pocketin which to place the pump. The incision was closed with a 5-0 prolene(Ethicon, Somerville, N.J.). Animal weights and tumor volumes (calipers)were recorded twice weekly. Volumes were calculated using the formulaD*d²*0.52 where D is the long diameter and d is the perpendicular shortdiameter. All animal experiments were approved by and performed inaccordance with the Institutional Animal Care and Use Committee of UTSouthwestern.

Immunohistochemistry. Formalin-fixed tissues were embedded in paraffin,sectioned and stained with hematoxylin and eosin by the MolecularHistopathology Laboratory at UT Southwestern. H&E photographs were takenat a total magnification of 40× on a Nikon DXM 1200 digital camera(Melville, N.Y.). For immunohistochemical staining, slides weredeparaffinized by heating at 60° C. for one hour followed by immersionin xylenes. Tissue was rehydrated by sequential immersion in ethanols.Endogenous peroxidase activity was blocked with 3% H₂O₂ in methanol.Antigen retrieval was performed using pH 6.0 citrate buffer (LabVision,Freemont, Calif.) for 15 minutes in a pressure cooker. Sections wereblocked with 20% Aquablock (EastCoast Bio, North Berwick, Me.) andsubsequently incubated overnight at 4° C. with 1:25 dilution of ratanti-mouse CD34 (ab8158, Abcam, Cambridge, Mass.). The primary antibodywas detected by incubating with a biotinylated donkey anti-rat IgG(Jackson ImmunoResearch, Westgrove, Pa.) followed by the ABC detectionkit (Vector laboratories, Burlingame, Calif.). Subsequent DAB(Invitrogen, Carlsbad, Calif.) detection was performed and slides werecounterstained with hematoxylin (Richard Allan Scientific, Kalamazoo,Mich.) and mounted with Crystal/Mount (Biomeda, Foster City, Calif.).Microvessel density (MVD) was determined by manually counting the numberof blood vessels in visible in the microscopic field of view (totalmagnification, 100×). Five fields were taken for each slide (n=3 pergroup).

B. Results

A cell-based binding screen for the isolation of VEGFR2-bindingpeptoids. The inventors have shown previously that combinatoriallibraries of peptoids (N-alkylglycine oligomers) are a rich source ofligands for many different proteins (Alluri et al., 2006; Alluri et al.,2003; Bachhawat-Sikder and Kodadek, 2003; Liu et al., 2005; Reddy etal., 2004). These experiments involved the synthesis of a “one bead-onecompound” library in which each bead displays many copies of a uniquepeptoid. The fluorescently-tagged protein target is introduced to thebead library in the presence of a large excess of unlabeled competitorproteins (i.e., in a crude lysate) and the few beads in the library thatretain the protein are identified by automated or manual detection ofthe most highly fluorescent beads. A significant drawback to applyingthis protocol to the isolation of peptoid ligands for integral membraneproteins such as VEGFR2 is that these receptors are often notwell-behaved biochemically. Various strategies have been introduced tocope with this problem, for example the expression of an isolatedextracellular domain or the incorporation of the full-length proteininto synthetic micelles or vesicles (Robelek et al., 2007). However, allof these approaches have well-documented drawbacks, and the inventorswished to develop a new type of assay that would allow the library to bescreened against the full-length receptor in as natural an environmentas possible.

Therefore, the inventors developed the assay shown in FIG. 1A. Porcineaortic endothelial (PAE/KDR) lacking the human VEGFR2 were labeled withgreen-emitting quantum dots and the same cells, but including a vectorthat directs expression of human VEGFR2, were labeled with red-emittingquantum dots. Note that the quantum dots were internalized into the celland thus should not affect their surface characteristics. In theorytherefore, the only difference between the two differentially labeledcell types should be the presence or absence of the target receptor. Ifa 1:1 mixture of the red- and green-labeled cells are incubated with thebead library, then beads that bind only red and not green cells would beexpected to display peptoids that are specific VEGFR2 ligands (FIG. 1A).

To execute this strategy, the inventors synthesized a library ofpeptoids with a theoretical diversity of approximately 250,000 compoundson TentaGel beads. The design of the library (FIG. 1B) attempted to takesome advantage of the structure of the VEGF-VEGR2 complex. The receptorconsists of seven extracellular immunoglobulin-like domains, a singletransmembrane region, and an intracellular tyrosine kinase domain(Matthews et al., 1991; Terman et al., 1991). VEGF binds toextracellular domains 2 and 3 (Fuh et al., 1998) of VEGFR2. It has beenreported that Isoleucine (Ile-43, 46, 83), Glutamate (Glu-64),Phenylalanine (Phe-17), Glutamine (Gln-79), Lysine (Lys-84) and Proline(Pro-85) side chains of VEGF are the most important moieties for thebinding to VEGFR2 (Muller et al., 1997). Therefore, in designing thispeptoid library, the inventors decided to have at least two of the aboveresidues fixed with the intent of biasing the library for VEGFR2binding. The inventors selected Lys-like and Leu-like residues to befixed at the C-terminus of each molecule in the library. Leu was choseninstead of Ile because Ile was not validated for peptoid synthesis atthe time of synthesis, and Leu was the most suitable substitute. Also,the inventors decided to place one additional Lys-like residue inbetween the resin and the 8-mer peptoid, making the full length of eachpeptoid nine residues. The positively charged Lys-like residue can repelthe peptoids from each other on the bead surface and would avoidaggregation of the peptoids that could hinder proper display. Thelibrary was synthesized on TentaGel macrobeads (140-170 μM diameter),which have excellent stability and swelling properties and also providea non-sticky surface that is ideal for reducing non-specific binding inscreening experiments (Alluri et al., 2003). Synthesis of the librarywas conducted using eight different amines (FIG. 1B) resulting in atheoretical diversity of 262,144 compounds.

The screen consisted of mixing a 1:1 ratio of the aforementioned red-and green-stained cells with the peptoid-displaying beads for 60-75 minat 37° C. After washing, the beads were examined under a fluorescencemicroscope with irradiation through a standard DAPI filter (FIGS. 1C and1D). When irradiated at this wavelength, the polystyrene-based beadsfluoresce blue whereas the quantum dots emit red and green light,respectively, allowing simultaneous visualization of the beads and boththe cells lacking and containing VEGFR2. Three screens, each using about100,000 beads, were conducted. As expected, in each case the vastmajority of the beads did not bind either green or red cells. A muchsmaller number were observed to bind both the red- and green-stainedcells approximately equally (large blue sphere with both green and redspeckles indicated with the arrow in FIG. 1C). These beads presumablydisplay peptoids that bind to molecules present on the surface of bothcell types. These were not characterized further. Only five of theapproximately 300,000 beads screened (approximately 0.0017% of thepopulation) were observed to bind red-stained cells only. One such beadis shown in FIG. 1D. These putative “hits” were collected using amicropipette and placed into separate tubes. They were then boiled in anSDS-containing solution to remove cells, proteins and other extraneousmatter and, after washing with aqueous buffer, subjected to automatedEdman degradation to determine their sequence. The deduced structures ofthe putative VEGFR2-binding peptoids are shown in FIG. 2.

The isolated peptoids are VEGFR2 ligands. Two of the ‘hits’ from thisscreen, GU40C and GU40E (see FIG. 2) were resynthesized, both as freecompounds and with C-terminal Cys (for C-terminal fluorescein taggingvia maleimide chemistry), cleaved from the beads and purified toapparent homogeneity by reverse phase HPLC. These compounds were choseninitially because they had the widest structural differences among theisolated peptoids and also inspection of their sequence suggested thatthey would be the most water-soluble peptoids. The association of thesepeptoids with VEGFR2 was evaluated using an ELISA-like assay. Acommercially available fusion protein containing the VEGFR2extracellular domain (amino acids 1-764 fused to 6× histidine-tagged Fcof human IgG, via the peptide IEGRMD; R&D Systems) was plated into96-well plates and then mixed with various concentrations ofN-terminally Fluorescein (FITC)-labeled peptoid. After washing awayunbound peptoid, the remaining fluorescence emission signal was measuredat 520 nm. In this assay purified VEGFR2 protein was used instead of thewhole PAE/KDR cells that over express VEGFR2, in order to confirm thesecompounds were binding only to the receptor.

As shown in FIG. 3A and Table 3, the peptoids were indeed found tobehave as specific VEGFR2 ligands with dissociation constants (KDs) ofabout 2 μM. These values are similar to (Zilberberg et al., 2003), orbetter than (Binetruy-Tournaire et al., 2000; Hetian et al., 2002) thosereported previously for peptidic VEGFR2 ligands. GU40C and GU40E did notbind detectably to CD105, another endothelial cell surface receptor, orGST, in the same assay (FIG. 3A). Conversely, a control peptoid notselected to bind VEGFR2 did not compete with GU40C or GU40E for bindingto VEGFR2 (FIG. 3B). Finally, if the immobilized receptor waspre-incubated with unlabeled VEGF, almost no binding of fluoresceinatedGU40C or GU40E could be observed, showing that these peptoids competefor the hormone for binding to VEGFR2 (not shown). The inventorsconclude that peptoids GU40C and GU40E are indeed specific ligands forhuman VEGFR2.

Functional characterization of the VEGFR2-binding peptoids. GU40C andGU40E were then tested for their ability to modulate VEGFR function. Ofcourse, since the screen simply demanded binding of the peptoid to thereceptor, it was not a given that this would be the case. An early stepin the angiogenesis cascade is auto phosphorylation of the VEGFR2intracellular kinase domain upon VEGF binding. In order to test whetherthe VEGFR2-binding peptoids could mimic or inhibit this VEGF-inducedVEGFR2 phosphorylation, the inventors incubated PAE/KDR cells that overexpress VEGFR2 with or without 1.3 nM VEGF and/or several differentconcentrations of the peptoid ligands. Western blot analysis usingphospho-VEGFR2-specific antibodies was then employed to monitor receptoractivation. Neither peptoid at any concentration tested evinced agonistactivity in the absence of VEGF.

Instead, both GU40C and GU40E appeared to be weak antagonists of VEGFdependent VEGR2 autophosphorylation. At a peptoid concentration of 500μM, an approximately 75% inhibition of phosphorylation was observed(FIG. 8). Note that that the hormone-receptor complex is extremelystable (K_(D)≈50 μM) (Fuh et al., 1998). Thus, the weak antagonisticactivity of these μM affinity peptoid ligands was not unexpected, butdoes highlight the requirement for a major increase in binding affinityif these molecules are to be of practical utility.

High Affinity Dimeric VEGR2-Binding Peptoids. Much of the high affinityof the VEGF-VEGR2 complex derives from the fact that the hormone andreceptor are both native dimers and associate in a 2:2 complex. Indeed,while the K_(D) of the (VEGF)₂. (VEGFR)₂ complex is 50 μM, a monomericderivative of VEGF binds to the VEGR2 receptor dimer with an affinity ofonly 1.5 μM (Fuh et al., 1998), a value quite similar to that exhibitedby the GU40C and GU40E peptoids. Therefore, the inventors hypothesizedthat a large increase in affinity could be achieved by joining twomolecules of either peptoid with an appropriate linker.

The VEGFR2 binding regions of the dimeric hormone are separated byapproximately 70 Å (Wiesmann et al., 1997). Therefore, the inventorssynthesized and characterized homodimeric derivatives of GU40C and GU40Ewith central Lys linker and additional linker arms that would place theN-termini of each peptoid between approximately 45-85 Å apart if thelinkers were in their fully conformation. This was accomplished by usinglinkers with different numbers and combinations of β-alanine andaminohexanoic acid units (FIG. 4A; Table 3, GU40C1-5 and GU40E1-5). Inaddition, shorter dimers were obtained for compound GU40C by avoidingthe additional linker region and also by truncating the three fixedC-terminal residues one at a time (FIG. 4A; Table 3, GU40CAC).

TABLE 3 Dimeric peptoid library design and their binding study resultsPeptoid Peptoid Structural features series name n1 n2 Nleu Nlys2 Nlys1K_(D) (nM) Fold improved GU40C GU40C — — Y Y Y 2854 ± 400 x GU40C1 N N YY Y 85.7 ± 4.0 33 GU40C2 2 N Y Y Y 268.7 ± 41.8 11 GU40C3 5 N Y Y Y 60.6 ± 23.4 47 GU40C4 2 5 Y Y Y 32.1 ± 4.9 89 GU40C5 5 5 Y Y Y 234.2 ±0.1  12 GU40CA N N Y Y N 97.5 ± 22  30 GU40CB N N Y N N weaker — GU40CCN N N N N weaker — GU40E GU40E — — Y Y Y 1213.1 ± 140   x GU40E1 N N Y YY 193.6 ± 21   6 GU40E2 2 N Y Y Y 591.1 ± 70   2 GU40E3 5 N Y Y Y 107.9± 9   11 GU40E4 2 5 Y Y Y 129.3 ± 12   9 GU40E5 5 5 Y Y Y 230 ± 39 5GU1040C1-5 and GU40E1-5 represent the longer dimers and GU40CA-C wereshorter dimers. In both longer and shorter dimers, two monomeric units(with or without linker region) were linked together via amide bondformations between C-terminal carboxylic acid moiety and each one of theamine groups on Lys side chain and N-terminus (FIG. 4A). Understructural features column, Y = present and N = absent of thecorresponding moiety. The values on n1 and n2 columns are the number ofcarbon atoms present in that region. K_(D) values are in nM and thefinal column show how many fold the binding was improved compared to themonomer in each case. ^(a)K_(D) values were determined by ELISA-likeassay.

The binding of each of these dimeric peptoids to VEGFR2 was analyzedusing the same ELISA-like method used for the monomeric ligands. Asshown in FIG. 4B and Table 3, all of the longer dimeric peptoidsexhibited improved affinity for the receptor, but the degree ofimprovement varied substantially depending on the nature of the linker.The best result was obtained with GU40C4 (FIG. 4C), which bound VEGFR2with an apparent KD of 32 μM (FIG. 4D), an improvement of about 90-foldrelative to the monomeric parent compound. This low nM dissociationconstant is similar to that exhibited by some monoclonal antibodies forVEGFR2 ((Brekken et al., 1998; Lu et al., 2002) even though thiscompound is far smaller than an antibody. Therefore, GU40C4 was chosenfor further study.

Specificity of the GU40C4-VEGFR2 interaction on the cell surface. Toevaluate the specificity of the high affinity dimeric peptoid forVEGFR2, various cell types were incubated with biotinylated GU40C4peptoid and association of the small molecule with the cells was probedusing streptavidin conjugated red quantum dots (Qtracker 655) (FIGS.5A-H). The DAPI-stained nuclei are shown in blue. GU40C4 binding toPAE/KDR cells that overexpress VEGFR2 was seen clearly (FIG. 5A), asexpected. This intense red signal disappeared completely in the presenceof 100-fold excess unlabeled GU40C4 (FIG. 5B). In addition, the red halowas not observed when the VEGFR2-expressing PAE/KDR cells were incubatedwith only the streptavidin-coated red quantum dots without addition ofthe biotinylated peptoid (FIG. 5C). Finally, biotinylated GU40C4 did notbind detectably to the PAE parental cells lacking the VEGFR2 expressionvector (FIG. 5D). Together, these data demonstrate that GU40C4 bindsspecifically to VEGFR2 and does not associate detectably with other cellsurface molecules displayed by the cell.

An important question is whether the peptoid is able to recognize VEGFR2on the surface of cells when the receptor is expressed at native levels.To address this question, the inventors incubated biotinylated CU40C4and streptavidin-coated red quantum dots with four different cell lines:HeLa, HEK-293, human foreskin fibroblast (HFF) and MCF-7. Of these, onlyMCF-7 expresses significant quantities of the VEGFR2. As seen in FIG.5D-5H, the red halo was observed only when the peptoid was incubatedwith the MCF-7 cells (see FIG. 5G). The inventors conclude from theseexperiments that the dimeric peptoid is highly specific for the VEGFR2and can recognize the receptor even when it is expressed at nativelevels on the surface of a breast cancer cell.

GU40C4 is a relatively potent antagonist of VEGF-dependent receptoractivation. Having demonstrated that GU40C4 is a high affinity and highspecificity ligand for the VEGFR2, the inventors next asked if it iscapable of antagonizing receptor function in vitro. To do so, theinventors examined the effect of the peptoid on VEGF-induced VEGFR2autophosphorylation by Western blotting, as described earlier. VEGFR2over-expressing PAE/KDR cells were induced with 1.3 nM VEGF in thepresence of the indicated concentrations of GU40C4 (FIG. 6A). Subsequentmeasurement of the fraction of VEGFR2 that had undergoneautophosphorylation demonstrated that the GU40C4 peptoid is indeed anantagonist of receptor activation with an IC₅₀ value of approximately0.9 μM (FIGS. 6A and 6B). This represents an improvement of between 100-to 500-fold over the monomeric parent compound (FIGS. 8 and 9),consistent with the increased affinity of the dimeric peptoid for thereceptor. Under the same conditions, 1 μM Avastin, the ultra-highaffinity anti-VEGF antibody employed clinically, completely blockedVEGFR2 autophosphorylation (FIGS. 6A and 6B).

The inventors also studied the ability of GU40C4 to inhibit VEGF-inducedproliferation of human vascular endothelial cells (HUVECs). HUVECs weregrown in 96-well plates and treated with 1.3 nM VEGF to induceproliferation. Various concentrations of GU40C4 were added as acompetitor and the effects were monitored after 4 days. As expected,GU40C4 was able to inhibit VEGF-induced HUVEC proliferation with an IC₅₀of approximately 1 μM. At 10 μM peptoid, cell proliferation was reducedto essentially basal levels (FIG. 6C). FLAG peptide (FIG. 10B), whichwas used as a negative control, did not show an effect on proliferationat 10 μM.

Finally, the inventors examined the effect of the GU40C4 on theVEGF-induced rearrangement of HUVECs into tubes in an endothelial cellmedium (ECM) gel, a commonly used in vitro model for VEGF-inducedangiogenesis. HUVECs were grown on ECM gel and treated with 1.3 nM VEGFand different concentrations of GU40C4 or various control compounds,including 10 μM unselected peptoid, GU40C (monomer) and GU40CC(ineffective shortest dimer; Table 3). Clear evidence of the inhibitionof tube formation could be seen at GU40C4 peptoid concentrations of 1 μMand 10 μM. The control peptoid and shortest dimer GU40CC with no bindinghad no effect. A slight effect was observed for monomeric GU40C which issmaller compared to its homodimer. From these experiments, it wasconcluded that the dimeric peptoid GU40C4 is a relatively potentantagonist of VEGFR2.

GU40C4 has in vivo therapeutic efficacy in a preclinical mouse model.Based on the in vitro data showing that GU40C4 blocked VEGF-mediatedeffects, the inventors hypothesized that the dimeric peptoid wouldinhibit tumor growth in a murine model. This was tested using A673(human Ewing's sarcoma) cells implanted into the flank of athymic nudemice. A total of 800 μg of GU40C4 (n=10) or a control peptoid (n=11) wasdelivered by an osmotic pump that was implanted subcutaneously at a sitedistant from the tumor on the day of tumor cell injection. The pumpseluted drug at a rate of 0.2 μl/hr continuously for a period of 20.8days. To validate the control peptoid, the inventors included a group ofanimals who received only saline (n=6) via the pump. Tumor growth in thesaline treated animals did not differ from tumor growth in controlpeptoid treated animals (data not shown). Importantly, since peptoidshave not been examined extensively in animals, we observed no adverseeffects of treatment with either GU40C4 or the control peptoid. Animalstreated with GU40C4 had a reduced tumor growth rate and significantlysmaller tumors at the end of therapy compared to control peptoid treatedanimals (FIG. 7A). On day 22 post tumor cell injection five animals fromeach group were sacrificed for histological analysis (FIGS. 7B-C). Theinventors continued to follow the remaining animals for ten days todetermine the extent of tumor growth delay in the GU40C4 treatedanimals. Within 3-4 days after cessation of therapy, tumors in theGU40C4 group began to grow at a rate similar to the control treatedanimals. However, tumors remained small for approximately one week,suggesting that GU40C4 retained some effect after delivery had stopped.Histological analysis revealed that GU40C4 reduced microvessel densityby approximately 50% at day 22 post tumor cell injection (p<0.0001, FIG.7B). Furthermore, GU40C4, but not the control peptoid, inducedsignificant tumor necrosis as evaluated by H&E histology (FIG. 7C). Thisis particularly striking when considering the smaller size ofGU40C4-treated tumors (approximately 200 mm³ at day 22) and the factthat A673 tumors of this size do not typically show evidence ofsubstantial necrosis. These data are consistent with GU40C4-mediatedinhibition of VEGF-induced angiogenesis in vivo and further validate ourin vitro studies.

C. Discussion

Many hormone-receptor interactions have been identified as therapeutictargets. The VEGF-VEGFR2 complex, in particular, has received muchattention given its essential role in angiogenesis. The majority of VEGFpathway inhibitors reported to date are either monoclonal antibodies(Brekken et al., 2000; Gerber et al., 2000; Kim et al., 1993; Prewett etal., 1999), other protein based molecules (Getmanova et al., 2006) orpeptides (D'Andrea et al., 2006) targeting VEGF itself or theextracellular domain of one of the VEGF receptors. The considerablesuccess of Avastin, a very high affinity anti-VEGF antibody highlightsthe utility of this strategy in the treatment of cancer and wet maculardegeneration, but also the limitations of therapeutic monoclonalantibodies. These must be injected or infused, have only limited tumorpenetrance and are difficult and costly to manufacture in largequantities. More classical small molecules are also used in this arena.Sutent (Sunitinib, Pfizer) (Motzer et al., 2006), Nexavar (Sorafenib,Bayer/Onyx) (Clark et al., 2005) and a few other small molecular VEGFreceptor tyrosine kinase (RTK)-targeted drugs have been approved foranti-angiogenic therapy. But due to the structural homology of manydifferent kinase domains, most of these inhibitors showcross-reactivity, decreasing the specificity of the drug (Fabian et al.,2005). Finally, as mentioned above, there are several reports of modestaffinity VEGF- and VEGFR2-binding peptides, but the limited serumhalf-life of these peptides limits their practical utility. Thus, thereremains a need for the development of new strategies with which totarget the hormone-receptor interaction itself with relatively lowmolecular weight molecules.

Isolation of receptor-binding peptoids as potential antibody surrogates.To circumvent the limitations of current technology mentioned above, theinventors sought to develop a general approach to the isolation ofreadily synthesized, protease-stable receptor-binding compounds. Theychose to employ a peptoid (oligo-N-substituted glycine) library as thestarting point. Peptoids have a number of favorable properties for thispotential application. They are even more straightforward to synthesizethan peptides (Figliozzi et al., 1996), yet are protease-insensitive(Simon et al., 1992). In addition, large collections of peptoids havebeen shown by us and others to be a rich source of protein-bindingligands (Kodadek et al., 2004). Finally, since the small amounts ofpeptoid on a single bead in a combinatorial library can be sequencedsensitively by Edman degradation (Alluri et al., 2003) or massspectrometry (Paulick et al., 2006), no encoding of the library isnecessary.

To simplify screening peptoid libraries for ligands to cell surfacereceptors, the inventors developed the two-color, cell-based screenshown in FIG. 1A. In this approach, the receptor target is introducedinto a cell type that does not normally express it. For example, humanVEGFR2 was over expressed in PAE cell in this study. These cells arelabeled with a red-emitting quantum dot. The parental line lacking thereceptor is labeled with green-emitting quantum dots and the cells arethen mixed and introduced to the bead-displayed peptoid library. Beadsthat bind only red cells and not green cells are then identified under afluorescence microscope and collected. After removing cells and otherdebris with a denaturing wash, the identities of the putativereceptor-binding peptoids are determined by Edman degradation.

This protocol avoids a common problem in targeting integral membranereceptors, which is the poor solubility and biochemical properties ofmany such proteins, often necessitating the use of detergents, micellesor other problematic reagents in screening experiments using recombinantreceptors. In this case, the receptor is displayed in a relativelynatural environment on the surface of the cell. Thus, this protocol isless likely to provide hits that do not bind the receptor under nativeconditions. It is important to point out that this is not the firststudy to employ cells as the targets in a bead-based screen. Inparticular, the elegant work of Lam and co-workers (Peng et al., 2006)is noteworthy. They reported a screen of an encoded-bead-based libraryof peptidomimetic compounds against α₄β₁ integrin that employedintegrin-expressing Jurkat cells. In this case however, the isolation ofa specific receptor ligand was dependent on the availability of a known,high-affinity receptor antagonist that could be used as a solublecompetitor in the screen. Otherwise most of the beads in the librarywere covered with cells. The use of the different colored cells that doand do not express the target receptor and the experimental conditionsthe inventors employed removes this limitation and allows this approachto be applied to almost any cell surface target.

It also means that a ligand for almost any accessible surface of thereceptor could be isolated, whereas in the competitive assay of Peng etal. (2006) only bead-displayed molecules that compete with the solublereceptor binding molecule will register. Of course, many cell-basedassays have been reported in which some easily monitored cellular eventdependent on receptor function, such as activation of a downstreamreporter gene, is triggered by a small molecule. But these assaysrequire spatial separation of the cells and molecules into differentwells of a microtiter plate and a significant robotics infrastructure tocarry out screens of large numbers of compounds. Furthermore, it isalways possible to isolate molecules that modulate the reporter event insome other way than by simply binding to the receptor. In contrast, thisscreen registers only selective binding to the target receptor andrequires no specialized equipment other than a fluorescence microscopeand can easily accommodate libraries containing hundreds of thousands ofmolecules. Thus, the inventors believe that the FIG. 1A assay representsa significant advance in receptor screening technology.

Dimeric peptoids as potent VEGFR2 antagonists. As reported above, ascreen of more than 250,000 peptoids with the general structure shown inFIG. 1B resulted in the isolation of five hits. Two were subsequentlyshown to be bona fide VEGFR2 ligands, while the other three remain to beanalyzed. Quantitative analysis revealed that the affinity of thesepeptoids for the receptor's extracellular domain was about 2 μM, a valuetypical for lead molecules isolated from peptoid libraries understandard conditions. Both peptoids proved to be low-potency antagonistsof the hormone-receptor interaction, though this was not demanded in thescreen. Since peptoids are peptide-like in their structure, theinventors speculate that the VEGF-binding surface of VEGR2 is a “hotspot” (Clackson and Wells, 1995; Mattos and Ringe, 1996) for bindingmolecules of this type, leading to a much greater likelihood ofisolating molecules that bind this surface. The poor potency ofantagonism was expected from the modest affinity of the peptoid and thevery high affinity of VEGF for VEGFR2.

To achieve higher affinity, the inventors used a simple dimerizationapproach that takes advantage of the fact that VEGFR2 is a native dimer,as is VEGF. Analysis of a small number of such compounds (FIG. 4 andTable 3) revealed that GU40C4 (FIG. 4C) bound VEGFR2 strongly, with adissociation constant of approximately 30 nM. A biotinylated version ofthis compound could be used to visualize the VEGFR2 on the surface ofnot only the cells used in the screen, but MCF-7 breast cancer cellsexpressing the receptor at the native level (FIG. 5). The peptoid didnot associate detectably with cells that lacked VEGFR2 on their surface(FIG. 5). This is an application for which one normally employs alabeled antibody and highlights the antibody-like binding properties ofthe peptoid. Yet its molecular mass is similar to that of only a21-residue peptide. In functional assays, peptoid GU40C4 proved to be areasonably potent inhibitor of VEGF-dependent VEGFR2autophosphorylation, endothelial cell proliferation and tube formationby HUVECs (FIGS. 6 and 7) with an IC₅₀ of approximately 1 μM. This is inthe range of what one would expect for a direct competition between thepeptoid and VEGF, given their respective affinities for the receptor(K_(D)s of 30 nM and 50 pm). Since GU40C is a completely unoptimizedlead compound, it should be possible to identify derivatives withsignificantly increased affinity for VEGFR2 and thus reduce the amountof compound necessary to compete the native hormone.

Example 4

Rapid identification of the ‘minimum pharmacophore’ of a lead compoundis a vital step in the drug development process since it sets the stagefor subsequent optimization. With peptide-based agents, this exercise issimplified by the regular structure of the molecule. A common practiceis to evaluate a series of derivatives in which each residue in turn isreplaced with a glycine or alanine (alanine scanning) (El Kasmi et al.,1998; Slon-Usakiewicz et al., 1997). Recently, the inventors reportedthe effective application of glycine scanning to a peptoid(N-substituted oligoglycine) inhibitor of the 19S regulatory particle ofthe proteasome. This allowed the inventors to create a minimalderivative of the original hit with about half the mass and thusincreased cell permeability and potency (Lim et al., 2008). They havealso reported the isolation of highly specific peptoid ligands for theextracellular domain of the Vascular Endothelial Growth FactorReceptor-2 (VEGFR2) (Udugamasooriya et al., 2008), an integral membranereceptor that triggers angiogenesis when bound by its cognate hormoneVEGF (Ferrara, 2004). A dimerized derivative (GU40C4) of one of thesenine residue peptoids (GU40C; see FIG. 13) is a low nM ligand for thereceptor's extracellular domain and is a potent antagonist ofangiogenesis in vivo (Udugamasooriya et al., 2008). Inhibition ofVEGFR2-mediated angiogenesis is a validated strategy to slow the growthof tumors as well as to treat “wet” macular degeneration (Ambresin andMantel, 2007; Brekken et al., 2000; D'Andrea et al., 2006; Gerber etal., 2000; Getmanova et al., 2006; Hicklin and Ellis, 2005; Hurwitz etal., 2004; Kim et al., 1993; Klohs and Hamby, 1999). Thus, this peptoidis of potential therapeutic interest and its optimization is animportant goal. Therefore, the inventors sought to identify the minimalpharmacophore in GU40C as the initial step in this effort.

First, nine derivatives of GU40C were synthesized in which each of thenine residues in the parent peptoid was replaced with a glycine. Allthese derivatives were synthesized with a C-terminal cysteine tofacilitate fluorescein attachment via maleimide chemistry. The affinityof each of these derivatives for the extracellular domain (ECD) ofVEGFR2 was then determined using an ELISA-like binding assay describedin the inventors' previous report (Udugamasooriya et al., 2008). Theresults are shown in FIG. 14 (black bars). Only two side chains (the6^(th) and 8^(th) from the C-terminus) appeared to be important forbinding of GU40C to the VEGFR2ECD. To buttress these data, the inventorsrepeated the analysis, but replaced each monomer in the peptoid withsarcosine rather than glycine. Since secondary amides have a strongpreference for a transoid configuration about the peptide bond, whiletertiary amides do not, it is possible that glycine substitution couldintroduce conformational constraints not present in the parent peptoidand thus the comparison of the derivative to the parent molecule mightreflect issues other than simply deleting the side chain. For example,if the preferred binding conformation of a peptoid involved a cisoidconformation about a particular peptide bond in the molecule, thenreplacement of the side chain with a hydrogen would discriminate againstthis conformation and presumably inhibit binding, even though the sidechain was not involved directly. A sarcosine scan has the effect ofreplacing each of the side chains in turn with a methyl group ratherthan a hydrogen, preserving the tertiary amide bond, but removing thebulk of the side chain. Therefore, the inventors decided to conduct asarcosine scan in the region of the molecule identified as beingcritical for binding by the glycine scan.

As shown in FIG. 14 (grey bars), substitution of the methyl group forisobutyl moiety at position 8 or the α-methylbenzyl group at position 6weakened binding of the peptoid for the VEGFR2 ECD significantly,consistent with the glycine scan results. However, in contrast with theglycine scanning result, substitution of the lysine-like side chain atposition 7 with methyl also reduced binding affinity. This result wasconfirmed by competition binding assays that compared directly therelative affinities of the peptoids with glycine and sarcosinesubstitution at position 7. The inventors do not fully understand thebasis of the different results obtained using the two scanning methodsat position 7. One possibility might be that a polar substituent capableof donating a hydrogen bond to solvent might be favorable there. In anycase, the combined data from the glycine and sarcosine scans indicatethat the N-terminal region of GU40C, specifically positions 6-8 (seeFIG. 13), are important for binding of the peptoid to VEGFR2.

Based on these data, it seemed reasonable to speculate that a trimericpeptoid including positions 6-8 would be a good ligand for the VEGFR2ECD, an interesting possibility since this molecule would have a mass ofless than 450 Daltons. To test this idea, the inventors synthesized afluorescein-conjugated tetramer peptoid GU40C(1) that contains the threeoriginal residues at 6-8 positions along with an N-terminal glycine(FIG. 15A).

The affinity of this minimized GU40C derivative for the receptor ECD wasthen tested, again using the ELISA-like binding assay. Somewhatsurprisingly, this molecule showed no detectable binding to the receptorECD at any of the concentrations tested (FIG. 15B). Combined with theglycine scanning data shown in FIG. 14, this result suggested thepossibility that some of the main chain atoms in the parent peptoidmight be involved in receptor binding.

Therefore, the inventors decided to reintroduce the full backbone, butleave out the side chains, except those important residues at positions6-8 (GU40C(2)-FIG. 16A, first structure). Interestingly, this compoundrecognized the receptor ECD with an affinity similar to that of theGU40C parent peptoid (FIG. 16B, square and circle data points),confirming the conclusion derived from the scanning experiments that theside chains at positions 1-5 are not involved in receptor binding. Thisobservation, combined with the sarcosine scanning data, confirm thatsome of the backbone amide bonds within the first five residuesparticipate in the binding event. The inventors also synthesized andtested GU40C(3) (FIG. 16A), a compound containing the essential sidechains and two amide groups C-terminal to these residues, but which areout of register with the amides in the parent peptoid. This compound hadno detectable affinity for the receptor ECD (FIG. 16B).

To identify the main chain amides important for binding, the inventorssynthesized five different truncated versions of GU40C, each containingall four of the N-terminal residues (FIG. 17A). Increasingconcentrations of these truncated versions were competed with a constantamount of fluoresceinated GU40C for binding to the VEGR2 ECD. Theresults are shown in FIG. 17B. Unlabeled GU40C competed efficiently withits labeled counterpart as expected (FIG. 17B). Elimination of the firstC-terminal residue weakened binding about ten-fold (FIG. 17B). Furtherelimination of the next three C-terminal residues further diminishedbinding only slightly. However, deletion of the next residue essentiallyabolished binding of the peptoid to the VEGFR2 ECD.

In summary, the data described above have defined the minimalpharmacophore of the peptoid VEGFR2 antagonist GU40C4. Only three sidechains are important for peptoid-receptor binding (the 6^(th) through8^(th) counting from the C-terminus; FIG. 18) as shown by glycine andsarcosine scanning (FIG. 14). However, a smaller peptoid containing onlythese residues is inactive (FIGS. 15A-B). Furthermore, an analysis oftruncated derivatives of GU40C showed that elimination of the firstC-terminal residue reduced the affinity of the peptoid by about ten-foldand removal of the fifth residue essentially abolished binding. Combinedwith the insensitivity of the binding affinity to the removal of theside chains at these residues, these data argue that it is the mainchain residues at positions 1 and 5 that contact the receptor ECD. Thismodel is further supported by the fact that the nine residue peptoidGU40C(2), which contains only the side chains at positions 6-8 but isotherwise comprised of glycines, binds the receptor ECD about as well asthe GU40C parent.

GU81 (FIG. 19) is the result of a medicinal chemistry exercise aimed atimproving the potency of the molecule. This derivative binds the VEGFR2about 4 times more tightly than the GU40C parent. The GU81 dimer (FIG.20) has a high affinity for the VEGFR2 and is a potent antagonist of itsactivity in vitro and in vivo. FIG. 21 shows the effect of doxorubicin(Dox) and the GU81 dimer (peptoid) on the growth of an aggressiveMMTV-induced tumor in a mouse. The data show that while the peptoidalone did not result in significant reduction in the rate of tumorgrowth, it did function synergistically with Dox. FIG. 22 shows theeffect of Dox and the GU81 dimer on the weight of the MMTV tumor asmeasured at the time of sacrifice (age 60 days). In agreement with themeasurement of tumor volume (see FIG. 21), the peptoid actssynergistically with Dox to reduce the growth of the tumor mass.

Example 5

The inventor sought to develop a rapid and sensitivity screen for agentsthat bind to pre-selected cell surface structures in a specific fashion.This assays was initially developed using cells expressing the VEGF-2receptor. Receptor-expressing cells or receptor-null cells were mixed inan approximately 1:1 ratio. The cells were then exposed to a library ofmolecules displayed on hydrophilic beads (FIG. 23) and after appropriateincubation and washing, the beads that bind only one color cell (red)were picked. The beads were boiled in SDS to remove the cells and otherdebris and the molecules bound are identified by automated Edmandegradation.

This two-color assay demands extremely high specificity, as if thebead-displayed molecule binds any other molecule on the cell surfaceother than the target receptor, then both colored cells will be retainedand the molecule will not be identified as a hit. As evidence of this,the inventors obtained only 5 hits out of 300,000 compounds using theVEGF-2 receptor as the target. Moreover, all of these molecules werelater shown to bind the same site on the receptor (Udugamasooriya etal., 2008).

Another example of this assay is shown in FIG. 25, where molecules thatbind specifically to autoimmune T cells were identified. Using thegeneral protocol shown in FIG. 23, CD4+ T cells isolated from an animalwith an autoimmune disease were labeled with red quantum dots, and CD4+T cells from a healthy control animal were labeled with green quantumdots. Note that unlike the experiment shown in FIG. 23, this screen isusing two different populations of cells. The hypothesis was that theautoimmune T cells involved in disease progression will be present inmuch higher levels in the autoimmune mouse than in the healthy control.Therefore, the bead library was screened for beads that bind only red Tcells (i.e., those isolated from the autoimmune animal). As shown inFIG. 25, when this experiment was performed using mice with EAE, anexperimental model of human multiple sclerosis, two peptoids that boundonly red cells were identified and sequenced. Subsequent follow-up work(data not shown) revealed that these peptoids also bound a clonal T cellpopulation known to bind the antigen used to initiate this autoimmunedisease (a peptide antigen from a nerve sheath protein), providingstrong evidence that these peptoids are true hits that recognizeautoimmune T cells.

The assay can be varied in a number of ways to achieve even morespecific results. For example, one can screen very large libraries formolecules that distinguish between closely related cell surfacereceptors. As shown in FIG. 24, the inventor sought to identify of apeptoid capable of binding to VEGFR2, but not VEGFR1. Therefore, alibrary was synthesized using differing side chains (500,000 compounds)and a three-cell (null, VEGFR2, VEGFR1), three-color screen wasperformed. This identified three peptoids (the structure of one is shownin FIG. 24) that bound only the VEGFR2-containing cells (labeled green)and not VEGFR1-containing cells (labeled orange) nor the receptor nullcells (labeled red).

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 4,680,338-   U.S. Pat. No. 5,141,648-   U.S. Pat. No. 5,440,013-   U.S. Pat. No. 5,446,128-   U.S. Pat. No. 5,475,085-   U.S. Pat. No. 5,563,250-   U.S. Pat. No. 5,618,914-   U.S. Pat. No. 5,670,155-   U.S. Pat. No. 5,672,681-   U.S. Pat. No. 5,674,976-   U.S. Pat. No. 5,710,245-   U.S. Pat. No. 5,840,833-   U.S. Pat. No. 5,856,456-   U.S. Pat. No. 5,859,184-   U.S. Pat. No. 5,880,270-   U.S. Pat. No. 5,929,237-   Alluri et al., J. Amer. Chem. Soc., 125:13995-14004, 2003.-   Alluri et al, Mol. BioSystems., 2:568-579, 2006.-   Ambresin and Mantel, Rev. Med. Suisse, 3:137-141, 2007.-   Bachhawat-Sikder and Kodadek, J. Amer. Chem. Soc., 125:9550-9551,    2003.-   Binetruy-Tournaire et al., EMBO J., 19:1525-1533, 2000.-   Brekken et al., Cancer Res., 58:1952-1959, 1998.-   Brekken et al., Cancer Res., 60:5117-5124, 2000.-   Cabebe and Wakelee, Drugs Today (Barc), 42:387-398, 2006.-   Ciardiello et al., Expert Opin. Emerg. Drugs, 8:501-514, 2003.-   Clackson and Wells, Science, 267:383-386, 1995.-   Clark et al., Clin. Cancer Res., 11:5472-5480, 2005.-   D'Andrea et al., Chem. Biol. Drug Des., 67:115-126, 2006.-   Dvorak, J. Clin. Oncol., 20:4368-4380, 2002.-   El Kasmi et al., Molec. Immunol., 35:905, 1998.-   Fabian et al., Nat. Biotechnol., 23:329-336, 2005.-   Ferrara, Endocrine Rev., 25:581, 2004.-   Figliozzi et al., Methods Enzymol., 267:437-447, 1996.-   Folkman, J. Natl. Cancer Inst., 82:4-6, 1990.-   Fuh et al., J. Biol. Chem., 273:11197-11204, 1998.-   Gerber and Ferrara, Cancer Res., 65:671-680, 2005.-   Gerber et al., Cancer Res., 60:6253-6258, 2000.-   Getmanova et al., Chem. Biol., 13:549-556, 2006.-   Hetian et al., J. Biol. Chem., 277:43137-43142, 2002.-   Hicklin and Ellis, J. Clin. Oncol., 23:1011-1027, 2005.-   Hurwitz et al., N. Engl. J. Med., 350:2335-2342, 2004.-   Johannesson et al. J. Med. Chem., 42(22):4524-4537, 1999.-   Johnson et al., In: Biotechnology And Pharmacy, Pezzuto et al.    (Eds.), Chapman and Hall, NY, 1993.-   Johnson et al., J. Clin. Oncol., 22:2184-2191, 2004.-   Kim et al., Nature, 362:841-844, 1993.-   Klohs and Hamby, Curr. Opin. Biotechnol., 10:544, 1999.-   Kodadek et al., Acc. Chem. Res., 37:711-718, 2004.-   Lim et al., Chem. Commun. (Cambridge, England), 1064, 2008.-   Liu et al., Curr. Pharm. Des., 13:143-162, 2007.-   Liu et al., J. Amer. Chem. Soc., 127:8254-8255, 2005.-   Lu et al., Int. J. Cancer, 97:393-399, 2002.-   Mateo et al., Hybridoma, 19:463-471, 2000.-   Matthews et al., Proc. Natl. Acad. Sci. USA, 88:9026-9030, 1991.-   Mattos and Ringe, Nature Biotechnol., 14:595-599, 1996.-   Millauer et al., Cell, 72:835-846, 1993.-   Motzer et al., J. Clin. Oncol., 24:16-24, 2006.-   Muller et al., Proc. Natl. Acad. Sci. USA, 94:7192-7197, 1997.-   Paulick et al., J. Comb. Chem., 8:417-426, 2006.-   Peng et al., Nature Chem. Biol., 2:381-389, 2006.-   Prewett et al., Cancer Res., 59:5209-5218, 1999.-   Reddy et al., Chem. Biol., 11:1127-1137, 2004.-   Remington's Pharmaceutical Sciences, 18^(th) Ed. Mack Printing    Company, 1990.-   Robelek et al., Angew Chem. Int. Ed. Engl., 46:605-608, 2007.-   Simon et al., Proc. Natl. Acad. Sci. USA, 89:9367-9371, 1992.-   Slon-Usakiewicz et al., Biochemistry, 36:3494, 1997.-   Stevenson, Leuk. Res., 29:239-246, 2005.-   Taylor, Intern. Med., 42:15-20, 2003.-   Terman et al., Oncogene, 6:1677-1683, 1991.-   Thomas, Cancer Nurs., 26:21S-25S, 2003.-   Udugamasooriya et al., J. Amer. Chem. Soc. 130, 5744-5752, 2008.-   Vita et al., Biopolymers, 47:93-100, 1998.-   Wawrzynczak & Thorpe, Cancer Treat Res., 37:239-251, 1988.-   Weisshoff et al., Eur. J. Biochem., 259(3):776-788, 1999.-   Whitty and Kumaravel, Nat. Chem. Biol., 2:112-118, 2006.-   Wiesmann et al., Cell, 91:695-704, 1997.-   Witte et al., Cancer Metastasis Rev., 17:155-161, 1998.-   Zeng et al., J. Biol. Chem., 276:26969-26979, 2001.-   Zilberberg et al., J. Biol. Chem., 278:35564-35573, 2003.-   Zuckermann et al., J. Amer. Chem. Soc., 114:10646-10647, 1992.-   Zuckermann et al., J. Med. Chem., 37:2678-2685, 1994.

1. A method of inhibiting VEFG signaling comprising contacting a cellexpressing a VEGFR2 with a compound having the formula:


2. The method of claim 1, wherein said compound is a dimer of theformula of claim 1, said dimer comprising a linker that replaces thefree —OH group of each monomer.
 3. The method of claim 1, wherein saidcell is an endothelial cell.
 4. The method of claim 3, wherein saidendothelial cell is a vascular endothelial cell.
 5. The method of claim1, wherein said cell is located in a human subject.
 6. The method ofclaim 5, wherein human subject suffers from glioma, sarcoma or myeloma.7. The method of claim 5, wherein human subject suffers from lungcancer, skill cancer, head & neck cancer, stomach cancer, breast cancer,colon cancer, pancreatic cancer, liver cancer, ovarian cancer, uterinecancer, cervical cancer, testicular cancer, rectal cancer, esophagealcancer, or brain cancer.
 8. The method of claim 1, wherein said compoundformulated in a lipid vehicle.
 9. The method of claim 6, wherein saidhuman subject is further treated with chemotherapeutic,radiotherapeutic, immunotherapeutic or anti-cancer gene therapy.
 10. Themethod of claim 7, wherein said human subject is further treated withchemotherapeutic, radiotherapeutic, immunotherapeutic or anti-cancergene therapy.
 11. The method of claim 5, wherein said human subjectsuffers from a non-cancer disease characterized by abnormal orpathologic angiogenesis.
 12. The method of claim 11, wherein saidnon-cancer disease is macular (wet) degeneration.
 13. The method ofclaim 12, wherein said human subject is further treated with a secondtherapy for said non-cancer disease state.
 14. The method of claim 5,wherein said human subject is contacted with said compound more thanonce.
 15. The method of claim 6, wherein said cancer is recurrent,metastatic, or multi-drug resistant.
 16. The method of claim 7, whereinsaid cancer is recurrent, metastatic, or multi-drug resistant.
 17. Apharmaceutical formulation comprising a compound having the structure:

dispersed in a pharmacologically acceptable medium, diluent orexcipient. 18-20. (canceled)
 21. A method of inhibiting VEFG signalingcomprising contacting a cell expressing a VEGFR2 with a compound havingthe formula:

wherein R is C₁₋₁₀₀₀, methyl, hydrogen, nitrogen, sulfur, oxygen,chlorine, bromine fluorine or silicon.
 22. The method of claim 21,wherein said compound is a dimer of the formula of claim 1, said dimercomprising a linker that replaces the free —OH group of each monomer.23. The method of claim 21, wherein said cell is an endothelial cell.24. The method of claim 23, wherein said endothelial cell is a vascularendothelial cell.
 25. The method of claim 21, wherein said cell islocated in a human subject.
 26. The method of claim 25, wherein humansubject suffers from glioma, sarcoma or myeloma.
 27. The method of claim25, wherein human subject suffers from lung cancer, skin cancer, head &neck cancer, stomach cancer, breast cancer, colon cancer, pancreaticcancer, liver cancer, ovarian cancer, uterine cancer, cervical cancer,testicular cancer, rectal cancer, esophageal cancer, or brain cancer.28. The method of claim 21, wherein said compound formulated in a lipidvehicle.
 29. The method of claim 26, wherein said human subject isfurther treated with chemotherapeutic, radiotherapeutic,immunotherapeutic or anti-cancer gene therapy.
 30. The method of claim27, wherein said human subject is further treated with chemotherapeutic,radiotherapeutic, immunotherapeutic or anti-cancer gene therapy.
 31. Themethod of claim 25, wherein said human subject suffers from a non-cancerdisease characterized by abnormal or pathologic angiogenesis.
 32. Themethod of claim 31, wherein said non-cancer disease is macular (wet)degeneration.
 33. The method of claim 32, wherein said human subject isfurther treated with a second therapy for said non-cancer disease state.34. The method of claim 25, wherein said human subject is contacted withsaid compound more than once.
 35. The method of claim 26, wherein saidcancer is recurrent, metastatic, or multi-drug resistant.
 36. The methodof claim 27, wherein said cancer is recurrent, metastatic, or multi-drugresistant.
 37. The method of claim 21, wherein the compound has thestructure:


38. The method of claim 21, wherein the compound has the structure:

wherein W is defined as C₁₋₁₀₀₀, hydrogen, nitrogen, sulfur, oxygen,chlorine, bromine, fluorine, silicon, biotin, or fluorescein.
 39. Apharmaceutical formulation comprising a compound having the structure:

and R is C₁₋₁₀₀₀, methyl, hydrogen, nitrogen, sulfur, oxygen, chlorine,bromine, fluorine or silicon, said compound being dispersed in apharmacologically acceptable medium, diluent or excipient. 40-62.(canceled)